Food additive comprising an amidase for detoxifying ochratoxin

ABSTRACT

The present invention relates to amidase enzymes and a feed or food additive comprising the amidase enzyme capable of degrading ochratoxin.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to PCT Application No. PCT/IB2011/053901 entitled “Food Additive Comprising An Amidase For Detoxifying Ochratoxin,” filed Sep. 6, 2011, which claims priority to EP No. 10177410.7, filed Sep. 17, 2010 and U.S. Provisional Application No. 61/380,280, filed Sep. 6, 2010, all of which are expressly incorporated by reference herein in their entirety.

SEQUENCE LISTING

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FIELD OF INVENTION

The present invention relates to methods of detoxifying mycotoxins. More specifically, the invention relates to amidase enzymes and feed and or food additives comprising at least one amidase for detoxifying ochratoxin, particularly ochratoxin A.

BACKGROUND

Mycotoxins are toxic secondary metabolites of fungi belonging, essentially, to the Aspergillus, Penicillium and Fusarium genera. They can be produced on a wide range of agricultural commodities and under a diverse range of agronomic, ecological and post harvest conditions worldwide.

Mycotoxins can enter the food chain in the field, during storage of a feed or food material, or at later points in the food chain. Their accumulation in foods and feeds represents a major threat to human and animal health since consumption of a mycotoxin-contaminated diet may result in teratogenic, carcinogenic and oestrogenic or immunosuppressive effects.

In 1985 the World Health Organization estimated that approximately 25% of the world's grains were contaminated by mycotoxins (Jelinek et al., 1989). This figure has likely grown since then due to an increase in global import and export of grains and cereals and the changing environmental and weather patterns.

Currently there are more than 400 mycotoxins documented but the mycotoxins of greatest concern and consequently the most studied include: aflatoxin, deoxynivalenol, zearalenone, fumonisin and ochratoxin.

Although there are many species of toxigenic moulds, only a few mycotoxins are considered to be significant for humans.

Three genera of fungi, Aspergillus, Penicillium, and Fusarium are most frequently involved with cases of mycotoxin contamination. Fungal colonization, growth and mycotoxin production are generally influenced by a variety of factors. The most important of which are temperature and water activity.

Generally, in warm regions aflatoxins are of major concern. This is because the Aspergillus species that produce these toxins find optimum conditions present in tropical regions. In contrast, Fusarium and Penicillium species have lower optimum temperatures and as a result are adapted to a more moderate climate. Ochratoxins, fumonisins and zearalenone are consequently produced in regions providing these conditions.

Ochratoxins are a group of mycotoxins produced as secondary metabolites by several fungi of the Aspergillus or Penicillium families and are weak organic acids consisting of a derivative of an isocoumarin. There are three generally recognized ochratoxins, designated A, B and C. Ochratoxin A is the most abundant member of the ochratoxin family and hence the most commonly detected, but is also the most toxic. Ochratoxin A (ochratoxin A) is a nephrotoxic, teratogenic, hepatotoxic, and carcinogenic mycotoxin present in cereals and other starch rich foods. Besides cereals and cereal products, ochratoxin A is also found in a range of other food commodities, including coffee, cocoa, wine, beer, pulses, spices, dried fruits, grape juice, pig kidney and other meat and meat products of non-ruminant animals exposed to feedstuffs contaminated with this mycotoxin. Many countries have set limits on ochratoxin A level in food, typically between 1 and 10 ppb (parts per billion) depending on the type and quality of the foodstuffs.

ochratoxin A production is due to a fungal infection in crops, in the field during growth, at harvest, in storage and in shipment under favourable environmental conditions, especially when they are not properly dried.

Ochratoxin A is a stable compound that can be hydrolysed into ochratoxin α (OTα) and L-phenylalanine by heating under reflux for 48 h in 6M hydrochloric acid (Van der Merwe et al., 1965) or with the carboxypeptidase A (Pitout, 1969). The conversion of OTA into ochratoxin α is considered to be a way to reduce its toxicity since OTα is commonly reported to be much less toxic than OTA. Moreover, ochratoxin α elimination half-time in the body (9.6 h) is shorter than that of OTA (103 h) (Li et al., 1997).

In order to ensure food safety, different approaches to prevent mycotoxin intake are developed at several stages along the food production chain.

It has been known since the 1970s that the mammalian digestive enzyme called carboxypeptidase A is cable of degrading OTA but that the efficiency of this enzyme is low. In fact in animals having carboxypeptidase A, such as pig, OTA toxicity due to its presence in feed is a problem. Furthermore, it has been found that OTA can to some extent inhibit carboxypeptidase A activity.

Prior to the present invention, there has been no disclosure of efficient enzyme solutions to degrade ochratoxins including ochratoxin A (OTA). Enzyme activities other than carboxypeptidase have been reported for OTA degradation, but until now no one has been able to identify a protein showing OTA degrading activity.

It is known that a commercial lipase product called “Amano™ lipase” which is a crude lipase produced from A. niger (Amano™ company, Japan) has ochratoxin degrading activity.

The OTA degrading activity in this lipase product has previously been attributed to a lipase or protease activity. For example, Maria A. Stander (J. Agric. Food Chem, 2000, 48, 5736-5739) concluded that the OTA degrading activity of Amano™ lipase resulted from a lipase.

Abrunhosa et al., (Biotechnology Lett., 2007, 29, 1909-1914) describe an enzyme preparation isolated from A. niger having OTA degrading activity. However, this enzyme preparation was not purified to an extent where the sequence of the active component could be determined at the amino acid or DNA level. Similar problems have been reported in other cases (Abrunhosa et al., TOXINS, [2010], 2, 1078-1099)

SUMMARY

The present invention is based on work undertaken by the inventors to identify and isolate an enzyme which is capable of efficiently degrading ochratoxin, more specifically ochratoxin A (OTA).

The inventors have discovered that contrary to their initial hypothesis formamidase from A. niger and A. nidulans and also the formamidase that exists in the Amano™ lipase product have no activity towards OTA but as expected do have activity towards formamide.

The inventors have surprisingly found that a 480 amino acid hypothetical protein, hereinafter referred to as amidase 2, which is encoded by an A. niger open reading frame has ochratoxin degrading activity, and particularly ochratoxin A degrading activity.

Furthermore, the inventors have also surprisingly found that the full length 480 amino acid amidase 2 comprising the N-terminal or signal sequence and having a MW of about 51 kDa (referred to as amidase 2 sig) has ochratoxin A degrading activity as well as the secreted mature 438 amino acid amidase 2 having a MW of about 47 kDa (referred to as amidase 2 mat).

It has been found that in amidase 2 mat, the N-terminal 42 amino acids are cleaved, that is, when amidase 2 is secreted into the culture medium its N-terminal 42 aa is cleaved to form the mature amidase 2 (i.e., amidase 2 mat) by an A. niger peptidase.

The inventors have isolated and cloned the A. niger gene encoding the amidase enzyme responsible for the degradation of ochratoxin A and have identified its crystal structure. They have identified that this gene encodes a polypeptide having the sequence of SEQ ID NO: 1 (amidase 2 sig). They have further identified that this enzyme is post translationally modified by cleavage of an N-terminal 42 amino acid sequence the mature amidase 2 shown as SEQ ID NO: 3

According to a first aspect of the present invention there is provided an isolated amidase enzyme capable of degrading ochratoxin. More particularly ochratoxin A

According to a second aspect of the present invention there is provided a food or feed additive comprising an amidase enzyme capable of degrading ochratoxin. More particularly ochratoxin A

According to a third aspect of the present invention there is provided a food or feed material comprising the feed additive of the present invention.

According to a Fourth aspect of the present invention there is provided a foodstuff or feedstuff comprising a feed material of the present invention.

According to a fifth aspect of the present invention there is provided a method for making a food or feed additive comprising admixing an amidase enzyme capable of degrading ochratoxin with at least one physiologically acceptable carrier.

According to a sixth aspect of the present invention there is provided a method of reducing ochratoxin contamination in a material comprising adding to said material an amidase enzyme capable of degrading ochratoxin.

According to a seventh aspect of the present invention there is provided a composition comprising an ochratoxin contaminated material and an amidase enzyme capable of degrading ochratoxin.

According to a eighth aspect of the present invention there is provided a method of making a foodstuff or feedstuff comprising adding to a food or feed material a food or feed additive according to the present invention.

According to a ninth aspect of the present invention there is provided a method of increasing the growth rate and/or health of an animal comprising feeding the animal an effective amount of a feedstuff according to the present invention.

According to an tenth aspect of the present invention there is provided the use of an amidase which degrades ochratoxin in the manufacture of a foodstuff or feedstuff.

According to a eleventh aspect of the present invention there is provided a foodstuff or feedstuff obtainable by the methods of the present invention.

According to a twelfth aspect of the present invention there is provided a recombinant cell comprising an amidase enzyme capable of degrading ochratoxin.

In a thirteenth aspect of the present invention there is provided an amidase enzyme comprising a polypeptide sequence having at least 30%, 35%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99% identity to SEQ ID NO:1 or SEQ ID NO: 3, or a polypeptide which differs from SEQ ID NO: 1 or SEQ ID NO: 3 by one or several amino acid additions, deletions and/or substitutions; or a polypeptide encoded by a polynucleotide having at least 30%, 35%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99% identity to SEQ ID NO:2, or a polynucleotide which differs from SEQ ID NO: 2 by one or several nucleotide additions, deletions and/or substitutions; and wherein said polypeptide is not SEQ ID NO:1.

According to a fourteenth aspect of the present invention there is provided a food or feed additive comprising the peptidase enzyme PEPAd (SEQ ID NO: 12) capable of degrading ochratoxin A.

According to a fifteenth aspect of the present invention there is provided a food or feed material comprising the feed additive of the thirteenth aspect.

According to a sixteenth aspect of the present invention there is provided a foodstuff or feedstuff comprising a feed material of the fourteenth aspect.

DETAILED DESCRIPTION OF THE INVENTION

In the description which follows, it will be understood that any of the preferred features described are applicable to any aspect of the present invention unless explicitly stated otherwise. It will be further understood that any of the preferred features are envisaged as being used in combination where appropriate.

It will be further understood that the terms ochratoxin or an amidase enzyme capable of degrading ochratoxin in preferred embodiments refers to ochratoxin A or an amidase enzyme capable of degrading ochratoxin A.

As used herein the term amidase (amidohydrolase) refers to an enzyme of the amidohydrolase superfamily which can hydrolyse an amide.

The inventors have isolated and cloned the amidase 2 enzyme from A. niger responsible for ochratoxin degradation. This enzyme is particularly effective at degrading ochratoxin A. They have shown that this enzyme is expressed by an ORF encoding a hypothetical 480 amino acid A. niger protein (An14g02080, Acc No. XP_(—)001400834) which shows ˜40% identity to certain bacterial amidases which are uncharacterised biochemically especially with respect to their substrate specificity.

Database searching indicates that this putative amidohydrolase (amidase 2) has 36% amino acid sequence identity with certain dipeptidases, such as the carboxypeptidase Sgx9355e encoded by an environmental DNA sequence isolated from the Sargasso Sea (Biochemistry 48 (2009): 4567-4576). The 3D structure of Sgx9355e is known and it is a member of the amidohydrolase superfamily (AHS). Sequence alignment of amidase 2 with Sgx9355e suggests that amidase 2 is as a member of the AHS.

This is a group of enzymes which have a remarkable substrate diversity with a (β/α)₈-barrel (Tim barrel) structural fold. Most of the enzymes within this superfamily catalyse the hydrolysis of C—O, C—N, or P—O bonds. No members of the superfamily have been linked to the hydrolysis of OTA.

It will be understood that the terms amidase (i.e amidohydrolase) and amidase which can degrade ochratoxin are used interchangeably herein unless specifically stated otherwise or it is obvious from the context that a different enzyme is being discussed.

The terms amidase and amidase which can degrade ochratoxin A refer to an enzyme which can break down at least 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 90%, 95%, 97%, 98%, 99% or 100% of the ochratoxin A present in a sample to ochratoxin α.

It will be apparent to the skilled person that any amidase which can degrade ochratoxin will be suitable for use in the present invention. Preferably, the amidase for use in any aspect of the present invention is an amidase which degrades at least ochratoxin A.

Preferably, the amidase enzymes according to the present invention comprises at least one, preferably, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, more preferably nine of the amino acid sequence motifs:

1) x-P-G-x-x-D-x-H-x-H-x-xG, where the two H is residues are in the active site;

2) G-x-T;

3) G-x-x-x-G-P;

4) G-H-x-D where the H is residue is in the active site;

5) D-G-x-x-x-C-x-x-x-x-R-x-x-x-R-x-x-A-x-x-I—K, where the Lys residue is in the active site

6) G-G-V-x-S-x-x-D-x-P, where the Val residue is in the active site;

7) V-x-A-H-x-x-G-x-x-G, where the two H is residues are in the active site;

8) H-x-x-x-x-D, where the H is residue is in the active site;

9) G-V-x-I-x-x-G-T-D, where Asp residue is in the active site.

Preferably, the motifs are:

1) x¹-P-G-x²-x³-D-x⁴-H-x⁵-H-x⁶-x⁷-G, where the two H is residues are in the active site;

2) G-x⁸-T;

3) G-x⁹-x ¹⁰-x¹¹-G-P;

4) G-H-x¹²-D where the H is residue is in the active site;

5)_(D-G-x) ¹³-x¹⁴-x¹⁵-C-x¹⁶-x¹⁷-x¹⁸-x¹⁹-R-x²⁰-x²¹-x²²-R-x²³-x²⁴-A-z²⁵-x²⁶-I-K, where the Lys residue is in the active site

6) G-G-V-x²⁷-S-x²⁸-x²⁹-D-x³⁹-P, where the Val residue is in the active site;

7) V-x³¹-A-H-x³²-x³³-G-x³⁴-x³⁵-G, where the two H is residues are in the active site;

8) H-x³⁶-x³⁷-x³⁸-x³⁹-D, where the H is residue is in the active site;

9) G-V-x⁴⁰-I-x⁴¹-x⁴²-G-T-D, where Asp residue is in the active site;

wherein:

x¹=l/m; x²=l/m; x³=w/i; x⁴=c/v/s/a; x⁵=any amino acid; x⁶=f/y/l; x⁷=any amino acid; x⁸=y/f; x⁹=t/a/s/v/r; x¹⁰=i/f/a; x¹¹=any amino acid; x¹²=g/s; x¹⁴=any amino acid; x¹⁵=e/d/g; x¹⁶=any amino acid; x¹⁷=any amino acid; x¹⁸=a/g/t; x¹⁹=v/a; x²⁰=any amino acid; x²¹=q/m/a; x²²=l/i/v; x²³=r/h/c; x²⁴=g/n; x²⁵=k/r/t/e/d; x²⁶=any amino acid; x²⁷=l/m/v/g; x²⁸=any amino acid; x²⁹=any amino acid; x³⁰=any amino acid; x³¹=a/s/h; x³²=c/v/a; x³³=h/q; x³⁴=k/r; x³⁵=any amino acid; x³⁶=g/v/a; x³⁷=s/t/i; x³⁸=y/f/e; x³⁹=l/a/i; x⁴⁰=any amino acid; x⁴¹=a/v; x⁴²=−l/a.

It will be understood that the term any amino acid refers to any one of amino acids G, A, V, L, I, M, F, W, P, S, T, C, Y, N, O, D, E, K, R or H, or an unnatural amino acid or amino acid derivative.

In a more preferred embodiment the motifs are:

1) l/m-P-G-l/m-w-D-c-H-x-H-f-x-G, where the two H is residues are in the active site;

2) G-y/f-T;

3) G-t-1-x-G-P;

4) G-H-g-D where the H is residue is in the active site;

5) D-G-v-x-e-C-x-x-a-v-R-x-q-l-R-r-g-A-k-x-l-K, where the Lys residue is in the active site

6) G-G-V-l/-S-x-x-D-x-P, where the Val residue is in the active site;

7) V-a-A-H-c-h-G-k-x-G, where the two H is residues are in the active site;

8) H-g-s-y-l-D, where the H is residue is in the active site;

9) G-V-x-l-a-l-G-T-D, where Asp residue is in the active site.

It will be readily apparent to the skilled person that the carboxypeptidase enzymes of the prior art which are capable of degrading ochratoxin do not comprise any of the recited motifs and show no sequence homology to amidases of the present invention.

In one preferred embodiment the amidase is an isolated amidase substantially free from other components in the culture media in which it is produced.

In a further preferred embodiment the amidase is at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 97%, 99% pure.

In a further preferred embodiment, the amidase is an amidase comprising a distorted Tim-like barrel structure including an active site comprising 6 histidine residues, 1 lysine residue and 1 aspartic acid residue, wherein the amino acids residues in the active site corresponding to positions H111, H113, H191, K246, H287, H289, H307 and D378 of SEQ ID NO:1 when the tertiary structure of the amidase and SEQ ID NO:1 are compared.

It will be understood by the skilled person that a Tim barrel is a conserved protein fold consisting of eight α-helices and eight parallel β-strands that alternate along the peptide backbone. Tim barrels are considered α/β protein folds because they include an alternating pattern of α-helices and β-strands in a single domain. In a Tim barrel the helices and strands (usually 8 of each) form a solenoid that curves around to close on itself in a doughnut shape, topologically known as a toroid. The parallel β-strands form the inner wall of the doughnut, whereas the α-helices form the outer wall of the doughnut. Each β-strand connects to the next adjacent strand in the barrel through a long right-handed loop that includes one of the helices.

The structure of amidase 2 can be divided into two domains, a core catalytic domain and a smaller β-sandwich domain. The catalytic domain comprises residues 107-425, which form a rather distorted TIM-like barrel of eight parallel β-strands (β5-7, 9-13) flanked on the outer face by α-helices. A kink in the middle of strand β7 occurring at residue 182 divides it into two separate strands, β7a and β7b. This kink represents the features that cause the barrel distortion. While β7a has only one neighbouring barrel strand, β6, strand β7b has two neighbours to which it is hydrogen-bonded, β8 and β9. The former represents a secondary structure element that is attached to the barrel core without formally being a part of it. The second cause for the barrel distortion is the absence of a canonical hydrogen-bonding interaction of residues 375-378 directly downstream of β13 with the first barrel strand β5 that would close the circle. Thus, the overall appearance of the domain core rather resembles a sandwich of parallel β-sheets, one consisting of three and the other of seven strands. Of them in total 13 helices (12 α and 1 g) of the catalytic domain those directly following the barrels strands and β8 can be assigned as barrel helices, while the remaining four helices represent additional secondary structure elements lining the top and bottom of the barrel.

The β-sandwich domain comprises residues of both the N- and the C-terminus (43-106, 426-480). The three strands formed by C-terminal residues (β14-16) are part of the larger of the two sheets. From the four strands formed by N-terminal residues β3 belongs to the smaller sheet, while the long and sharply bent strands β1, β2 and β4 contribute to the formation of both sheets. Only β1 and β4 are parallel to each other. Helical turns γ16 and γ17 are inserted between β14 and β15 and pack against the catalytic domain. Helices γ1 and α2 inserted between β3 and β4 are pointing in the opposite direction and are solvent exposed.

It will be understood by the skilled person that the tertiary structure of a given amidase can be readily compared to the tertiary structure of SEQ ID NO:1 having the co-ordinates shown in FIG. 25 using standard software packages with their default parameters, for example, PyMOL (www. Pymol.org).

It will be further readily apparent to the skilled person that the carboxypeptidase enzymes of the prior art which are capable of degrading ochratoxin do not comprise a Tim barrel-like structure including the specified residues in the active site.

In one embodiment, the amidase is an enzyme of EC 3.5.1.X, according to the Recommendations of the Nomenclature Committee of the International Union of Biochemistry and Molecular Biology (IUBMB) where X is a designation number provided by the committee.

It will be understood by the skilled person that carboxypeptidase A and carboxypeptidase Y of the prior art are designated in EC 3.4.17.1 and 3.4.16.5 respectively.

In one preferred embodiment, the amidase is one which degrades ochratoxin A when incubated at pH3-9 and 21-40° C.

It will be apparent that the ability of an amidase enzyme to degrade ochratoxin A can be determined using the OTA degradation assay described in the method section below.

Suitable amidases comprise those from Aspergillus niger, Aspergillus flavus, Talaromyces stipitatus, Neurospora crassa, Streptomyces, for example, S. roseosporus, Thermotoga lettingae, Salinispora arenicola, Glomerella graminicola, Metarhizium anisopliae and Aspergillus oryzae shown as SEQ ID NO: 1, 3, 5, 6, 7, 8, 9, 10, 11, 13, 14 and 15 respectively or a sequence having at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 90%, 95%, 98%, 99% identity to any one of SEQ ID NOs:1, 3, 5 to 11, 13, 14 or 15.

In preferred embodiments of the present invention the amidase enzyme for use in the additives, foods, feeds, uses and methods of the present invention comprises a polypeptide having the sequence of SEQ ID NO: 1 or SEQ ID NO: 3, or a sequence having at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 90%, 95%, 98%, 99% identity to SEQ ID NO: 1 or SEQ ID NO: 3, or a polypeptide which differs from SEQ ID NO: 1 or SEQ ID NO 3 by one or several amino acid additions, deletions and/or substitutions; or a polypeptide produced by expression of a polynucleotide comprising SEQ ID NO: 2 or a polynucleotide having at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 90%, 95%, 98%, 99% identity to SEQ ID NO:2, or a polynucleotide which differs from SEQ ID NO: 2 by one or several nucleotide additions, deletions and/or substitutions; or a sequence which hybridises under stringent conditions to the complement of SEQ ID NO:2, or a sequence having at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 90%, 95%, 97%, 98% or 99% identity thereto.

In further preferred embodiments, the enzyme according to the present invention catalyzes the chemical bond breakage of C—N, C—O or P—O, more preferably C—N.

It will be apparent to the skilled person that full length amidase 2 variants can be made using any well known technique in the art. More stable variants resistant to cleavage of the N-terminal or signal sequence can be produced by mutating the protease liable peptide bonds in the peptide sequence.

For example, mutations can be made at one or more of the following 71 sites of the 480aa amidase 2 sequence shown in SEC) ID NO: 1 these may alter the sensitivity to hydrolysis by signal peptidases, peptidases secreted by the host organism into its medium and pepsin:

18 31 33 34 42 55 56 56 64 69 70 78 79 107 114 127 128 139 146 147 156 184 185 194 195 197 202 203 220 236 262 264 269 270 304 317 318 325 335 336 336 337 343 344 351 352 354 355 359 360 374 375 386 387 388 389 390 391 400 412 420 424 425 435 436 440 446 461 462 462 480;

mutations at the following 44 sites may alter trypsin resistance:

3 4 36 48 66 75 88 89 92 99 138 141 155 166 231 232 235 239 240 243 246 256 271 279 282 291 299 303 320 322 330 346 351 361 368 395 405 426 445 451 458 461 464 476;

mutations at the following 90 sites may alter chymotrypsin resistance: 1 11 16 19 23 32 34 39 53 56 57 65 70 79 90 91 98 103 107 108 112 113 114 120 121 124 128 139 144 147 151 154 157 160 179 185 191 195 203 206 209 215 216 221 236 248 254 262 265 270 287 289 295 305 307 310 316 318 319 325 326 336 337 344 349 352 355 359 360 363 375 387 389 391 398 401 413 421 425 429 436 441 447 455 457 462 463 471 479 480.

It will be further understood that the 480 aa amidase 2 polypeptide or its variants may be N- and/or O-glycosylated at any of its asparagine (Asn), serine (Ser) and threonine (Thr) residues in order to improve solubility and heat-stability.

It will be understood that as defined herein, the term stringent conditions refers to washing at. 50° C. and 0.2×SSC {1×SSC=0.15 M NaCl, 0.015 M Na₃citrate pH 7.0}.

It will be understood that these conditions may also be high stringent conditions which are defined herein as washing at 65° C. and 0.1×SSC {1×SSC=0.15 M NaCl, 0.015 M Na₃citrate pH 7.0}.

In another preferred embodiment, the amidase enzyme is produced by expression of a polynucleotide encoding a polypeptide having an amino acid sequence comprising SEQ ID NO: 1, 3, 5-11, 13, 14 or 15, or a sequence having at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 90%, 95%, 97%, 98%, 99% identity thereto, or a polypeptide which differs from SEQ ID NO: 1, 3, 5-11, 13, 14 or 15 by one or several amino acid additions, deletions and/or substitutions.

More preferably, the amidase is encoded by a polynucleotide selected from:—

-   -   a) a polynucleotide comprising SEQ ID NO: 2 or a sequence having         at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%,         99% identity thereto, or a polynucleotide which differs from SEQ         ID NO: 2 by one or several nucleotide additions, deletions         and/or substitutions;     -   b) a polynucleotide which differs from SEQ ID NO: 2 due to the         degeneracy of the genetic code;     -   c) a polynucleotide which hybridises under stringent conditions         to the complement of SEQ ID NO: 2 or a sequence having at least         10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%         identity thereto, or a polynucleotide which differs from the         complement of SEQ ID NO: 2 by one or several nucleotide         additions, deletions and/or substitutions.

In another preferred embodiment, there is provided a vector comprising a polynucleotide encoding the amidase enzyme which degrades ochratoxin A for use in the present invention.

It will be apparent to the skilled person that the vector can be any suitable expression vector and that the choice of vector may vary depending upon the type of cell into which the vector is to be inserted. Suitable vectors include pGAPT-PG, pRAX1, pGAMD and pGPT-pyrG1.

In a further preferred embodiment, the vector is comprised in a cell. In a further embodiment, the cell is a spore.

It will be understood that as used herein the term spore refers to a fungal or bacterial spore, endospore or exospore.

The cell according to the present invention may be any suitable cell. More preferably, any suitable bacterial, fungal or plant cell. Even more preferably the cell is selected from E. coli, Streptomyces, Hansenula, Trichoderma (particularly T. reesei), Bacillus, Lactobacillus, Aspergillus (particularly A. niger), a plant cell and/or spores of Bacillus, Trichoderma, or Aspergillus.

In a more preferred embodiment there is provided a recombinant cell or spore comprising a polynucleotide encoding an amidase enzyme which degrades ochratoxin, preferably ochratoxin A for use in the present invention.

In further preferred embodiments, the amidase enzyme for use in the present invention is recombinant.

In a preferred aspect of the present invention there is provided a food or feed additive comprising an amidase enzyme which is capable of degrading ochratoxin.

Preferably, the amidase enzyme will degrade at least ochratoxin A.

More preferably, the amidase enzyme will also degrade at least one other ochratoxin, more preferably, at, least ochratoxin B.

In a further embodiment, the enzyme will also degrade at least one ochratoxin derivatives beside ochratoxin B and ochratoxin C, and/or at least one ergot alkaloid.

It will be understood that ergot alkaloids are compounds containing amide bonds and include, for example, ergocornine, ergocorninine, ergocristine, ergocristinine, ergocryptine, ergocryptinine, ergometrine, ergosine, ergotamine and ergotaminine. These compounds are toxic to living organisms including humans and farm animals.

It will be apparent to the skilled person that this food or feed additive may be added to a food or feed material contaminated with ochratoxin in order to reduce the level of the toxin present in the food or feedstuff consumed by an animal. Ochratoxin A is known to be an important contaminant of cereals and other starch rich foods as well as, for example, coffee, cocoa, wine, beer, pulses, spices, dried fruit, grape juice, milk and meat products from non ruminant animals. Ochratoxin A is a nephrotoxic, teratogenic, hepatotoxic and carcinogenic compound which if present even at low levels can be harmful.

In a more preferred embodiment the additive is a feed additive.

In preferred embodiments the food or feed additive comprises the amidase at a level of at least 0.001 g/kg, at least 0.01 g/kg, at least 0.1 g/kg, at least 1 g/kg, at least 5 g/kg, at least 7.5 g/kg, at least 10.0 g/kg, at least 15.0 g/kg, at least 20.0 g/kg, at least 25.0 g/kg. Preferably, the food or feed additive comprises the amidase at a level such that when added to a food or feed material, the feed material comprises the amidase in a range of 1-500 mg/kg, 1-100 mg/kg, more preferably 2-50 mg/kg or 2-10 mg/kg.

In preferred embodiments of the present invention the amidase can hydrolyse at least 10, 20, 50, 100, 200, 300, 500, 700, 900, 1000 nanomoles OTA per min per mg protein at pH 7.0 and 40° C. when the OTA is present at a concentration of 1 μg/ml.

More preferably, the food or feed additive of the present invention comprises a recombinant cell capable of expressing an amidase enzyme capable of degrading ochratoxin. More preferably, the amidase enzyme is capable of degrading at least ochratoxin A.

Even more preferably, the amidase enzyme is also capable of degrading at least one further ochratoxin or ochratoxin derivative and/or at least one ergot alkaloid, more preferably, ochratoxin B.

Most preferably, the amidase comprises SEQ ID NO: 1, SEQ ID NO: 3 or any one of SEQ ID NO: 5-11, 13, 14 or 15 or a sequence having at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 90%, 95%, 98%, 99% identity to any one of SEQ ID NOs:1, 3, 5 to 11, 13, 14 or 15.

In another preferred embodiment the cell is an Aspergillus cell or spore. It will be apparent to the skilled person that Aspergillus cell may be a living cell, a dead cell or a disrupted cell.

In preferred embodiments, the recombinant cell is an A. niger cell. More preferably, the cell has increased OTA degrading activity when compared to a non recombinant cell of the same species.

It will be understood by the skilled person that the amidase enzyme capable of degrading ochratoxin A for use in the present invention can be provided independently as either liquid or solid/granulated compositions.

Preferably, when said enzyme is in liquid form, said enzyme is secreted into the medium following culturing of a cell comprising said enzyme. Preferably said medium is cell-free (i.e. the cell(s) have been separated from the medium). Preferably said medium is concentrated. It will be understood that the medium can be granulated to provide a solid enzyme composition.

It will be further understood that the food or feed additive according to the present invention may be provided in the form of a solution or as a solid—depending on the use and/or the mode of application and/or the mode of administration.

In a further embodiment, the additive may be used to pre-treat a material which will be used as a food or feed.

For example, the amidase may be used for treating liquids such as those produced as by-products from ethanol plants. In this case, the additive will be added to a liquid fermentation broth in order to degrade ochratoxin A contaminants found in the medium used to grow yeast or microbes for ethanol production.

It will be understood that the broth may be subsequently dried and fed to animals.

In an alternativel embodiment, the additive may be added to milk, for example, cows milk, contaminated with OTA.

In one embodiment the food or feed additive according to the present invention is in a liquid formulation suitable for consumption, preferably such liquid composition further comprises at least one of a buffer, a salt, sorbitol and/or glycerol.

Preferably, the food or feed additive further comprises at least one physiologically acceptable carrier.

The physiologically acceptable carrier is preferably selected from at least one of maltodextrin, limestone (calcium carbonate), cyclodextrin, wheat or a wheat component, sucrose, starch, Na₂SO₄, Talc, PVA and mixtures thereof.

In a further embodiment, the food or feed additive may further comprise a metal ion chelator. The metal ion chelator may be selected from EDTA or citric acid.

In one embodiment the amidase enzyme is dried on the physiologically acceptable carrier.

In one embodiment the food or feed additive is granulated or co-granulated with other enzymes.

In preferred embodiments, the amidase for use in the present invention may be used is combination with one or more further enzyme. In preferred embodiments, the one or more further enzyme is selected from the group consisting of those involved in protein degradation including carboxypeptidases preferably carboxypeptidase A, carboxypeptidase Y, A. niger aspartic acid proteases of PEPAa, PEPAb, PEPAc and PEPAd, elastase, amino peptidases, pepsin or pepsin-like, trypsin or trypsin—like proteases and bacterial proteases including subtilisin and its variants, and of those involved in starch metabolism, fibre degradation, lipid metabolism, proteins or enzymes involved in glycogen metabolism, enzymes which degrade other contaminants, acetyl esterases, amylases, arabinases, arabinofuranosidases, exo- and endo-peptidases, catalases, cellulases, chitinases, chymosin, cutinase, deoxyribonucleases, epimerases, esterases, formamidase, -galactosidases, for example α or β-galactosidases, exo-glucanases, glucan lysases, endo-glucanases, glucoamylases, glucose oxidases, -glucosidases, for example α or β-glucosidases, glucuronidases, hemicellulases, hydrolases, invertases, isomerases, laccases, phenol oxidases, lipase, lyases, mannosidases, oxidases, oxidoreductases, pectinase, pectate lyases, pectin acetyl esterases, pectin depolymerases, peptidase, pectin methyl esterases, pectinolytic enzymes, peroxidases, phenoloxidases, phytase, polygalacturonases, rhamno-galacturonases, ribonucleases, thaumatin, transferases, transport proteins, transglutaminases, xylanases, hexose oxidase (D-hexose: O₂-oxidoreductase, EC 1.1.3.5), acid phosphatases and/or others or combinations thereof. These include enzymes that, for example, modulate the viscosity of the composition or feed.

In a particularly preferred embodiment, the enzyme is used in combination with carboxypeptidase A.

In a further preferred embodiment, the enzyme is used in combination with PEPAd protease.

In a further embodiment the amidase may be used in combination with at least one of a detoxifying enzyme selected from the group consisting of a mycotoxin degrading enzyme, for example, aflatoxin detoxizyme, zearalenone esterases, zearalenone lactonases, fumonisin carboxylesterases, fumonisin aminotransferases, aminopolyol amine oxidases, deoxynivalenol expoxide hydrolases, a mycotoxin degrading microorganism, for example, Bacillus subtilis, B. licheniformis, Lactobacillus or an absorbent (i.e., myctoxin binders) including at least one polymer, for example, microbial cell walls or an inorganic material such as bentonite.

It will be understood that the feed additive may be for any suitable animal. In one preferred embodiment the animal is a monogastric animal, for example, poultry, swine, fish, shellfish and crustacea, for example, shrimps, pet animals such as, for example, cats or dogs. In an alternative preferred example the animal is a ruminant selected from, for example, cows or other bovines, sheep, goats, camels, deer, llama, antelope, alpacas or wildebeest.

It will be obvious to the skilled person that the food or feed additive according to the present invention may also comprise other components such as stabilising agents and/or bulking agents and/or other enzymes.

Preferably, the food or feed additive according to the present invention will be thermally stable to heat treatment up to about 70° C.; up to about 80° C.; or up to about 95° C. The heat treatment may be performed for up to about 0.5 minute; up to about 5 minutes; up to about 10 minutes; up to about 30 minutes; up to about 60 minutes. The term thermally stable means that at least about 75% of the enzyme components that were present/active in the additive before heating to the specified temperature are still present/active after it cools to room temperature. Preferably, at least about 80% of the enzyme components that were present and active in the additive before heating to the specified temperature are still present and active after it cools to room temperature.

The food or feed additive according to the present invention may have a shelf-life of greater than about 30 weeks; greater than about 40 weeks; greater than about 50 weeks; greater than about 1 year; greater than about 1.5 years. The shelf-life means that at least about 80% of the enzyme components that were present and active in the additive when it was prepared are still present and active.

Preferably, the method of preparing a food or feed additive according to the present invention comprises a mixing step that comprises admixing the amidase enzyme which degrades ochratoxin, preferably ochratoxin A, optionally with at least one physiologically acceptable carrier.

More preferably, the amidase enzyme is also capable of degrading at least one further ochratoxin or ochratoxin derivative and/or at least one ergot alkaloid, more preferably, ochratoxin B.

In a particularly preferred embodiment the food or feed additive is homogenized to produce a powder

In an alternative preferred embodiment, the food or feed additive is formulated in to granules as described in WO20071044968 (referred to as TPT granules) incorporated herein by reference.

In another preferred embodiment when the food or feed additive is formulated into granules the granules comprises a hydrated barrier salt coated over the protein core. The advantage of such salt coating is improved thermo-tolerance, improved storage stability and protection against other food or feed additives otherwise having adverse effect on the enzyme.

Preferably, the salt used for the salt coating has a water activity less than 0.25 or constant humidity greater than 60% at 20° C.

Preferably, the salt coating comprises a Na₂SO₄.

The method of preparing a food or feed additive may also comprise the further step of pelleting the powder. The powder may be mixed with other components known in the art. The powder, or mixture comprising the powder, may be forced through a die and the resulting strands are cut into suitable pellets of variable length.

Optionally, the pelleting step may include a steam treatment, or conditioning stage, prior to formation of the pellets. The mixture comprising the powder may be placed in a conditioner, e.g. a mixer with steam injection. The mixture is heated in the conditioner up to a specified temperature, such as from 60-100° C., typical temperatures would be 70° C., 85° C., 90° C. or 95° C. The residence time can be variable from seconds to minutes and even hours. Such as 5 seconds, 10 seconds, 15 seconds, 30 seconds, 1 minutes 2 minutes., 5 minutes, 10 minutes, 15 minutes, 30 minutes and 1 hour.

In a further aspect there is provided a food or feed material comprising the food or feed additive of the present invention. It will be understood that the food or feed additive of the present invention is suitable for addition to any appropriate food or feed material.

It will be obvious to the skilled person that the food or feed additive can be added to any food or feed material as a precautionary step. Alternatively, the food or feed additive can be added to food or feed materials which are known to be prone to ochratoxin, preferably ochratoxin A, contamination or to food or feed materials which have been shown to be contaminated with ochratoxin, preferably ochratoxin A. It will be further apparent to the skilled person that the presence of ochratoxin, preferably ochratoxin A can be identified by any suitable means, for example, HPLC, ELISA or through the use of commercially available ochratoxin detection strips (Helica Biosystems, Inc., Fullerton Calif.).

As used herein, the term feed material refers to the basic feed material to be consumed by an animal. It will be further understood that this may comprise, for example, at least one or more unprocessed grains, and/or processed plant and/or animal material such as soybean meal or bone meal.

In some embodiments, the feed material will comprise one or more of the following components: a) cereals, such as small grains (e.g., wheat, barley, rye, oats and combinations thereof) and/or large grains such as maize or sorghum; b) by products from cereals, such as corn gluten meal, Distillers Dried Grain Solubles (DDGS), wheat bran, wheat middlings, wheat shorts, rice bran, rice hulls, oat hulls, palm kernel, and citrus pulp; c) silage such as maize silage; d) protein obtained from sources such as soya, sunflower, peanut, lupin, peas, fava beans, cotton, canola, fish meal, dried plasma protein, meat and bone meal, potato protein, whey, copra, sesame; e) oils and fats obtained from vegetable and animal sources; f) minerals and vitamins.

As used herein, the term feedstuff refers to a feed material to which one or more feed additives have been added. According to another aspect there is provided a feedstuff comprising the feed material of the present invention.

It will be understood by the skilled person that different animals require different feedstuffs, and even the same animal may require different feedstuffs, depending upon the purpose for which the animal is reared.

Preferably, the feedstuff may comprise feed materials comprising maize or corn, wheat, barley, triticale, rye, rice, tapioca, sorghum, and/or any of the by-products, as well as protein rich components like soybean mean, rape seed meal, canola meal, cotton seed meal, sunflower seed mean, animal-by-product meals and mixtures thereof. More preferably, the feedstuff may comprise animal fats and/or vegetable oils.

Optionally, the feedstuff may also contain additional minerals such as, for example, calcium and/or additional vitamins.

As used herein, the term food material refers to a basic food to be consumed by a human. It will be understood that this may comprise grain, plant or animal material.

In some embodiments, the food material may comprise one or more of a cereal or cereal product, coffee, cocoa, wine, beer, pulses, spices, dried fruits, grape juice, milk, meat or meat products.

As used herein, the term foodstuff refers to a food material to which one or more food additives have been added. According to another aspect there is provided a foodstuff comprising the food material of the present invention.

In preferred embodiments, the foodstuff or feedstuff comprises amidase at a level of about 0.001 mg-10 g/kg, 0.01 mg-10 g/kg, 0.1 mg-10 g/kg, 0.1 mg-5 g/kg, 1 mg-5 g/kg, 0.5 g-1 g/kg food/feedstuff.

It will be readily apparent to the skilled person that in order for the food or feed additive of the present invention to provide the claimed advantages the food/feedstuff must be a food/feedstuff contaminated with ochratoxin, preferably ochratoxin A.

In another aspect there is provided a method for producing a feedstuff. Feedstuff is typically produced in feed mills in which raw materials are first ground to a suitable particle size and then mixed with appropriate additives. The feedstuff may then be produced as a mash or pellets; the later typically involves a method by which the temperature is raised to a target level and then the feed is passed through a die to produce pellets of a particular size. The pellets are allowed to cool. Subsequently liquid additives such as fat and enzyme may be added. Production of feedstuff may also involve an additional step that includes extrusion or expansion prior to pelleting—in particular by suitable techniques that may include at least the use of steam.

The feedstuff may be a feedstuff for any suitable animal. Preferably, the feedstuff is for domestic or farm animals.

In one embodiment the animal is a monogastric animal, such as poultry (for example, broiler, layer, broiler breeders, turkey, duck, geese, water fowl), swine (all age categories), fish, shell fish including crustaceans such as shrimps, a pet (for example dogs, cats).

In a further embodiment the animal is a ruminant, such as a bovine (for example, cow water buffalo, bison, yak), sheep, goats, camels, deer, llamas, antelope, alpacas or wildebeest.

The feedstuff may comprise at least 0.0001% by weight of the feed additive. Suitably, the feedstuff may comprise at least 0.0005%; at least 0.0010%; at least 0.0020%; at least 0.0025%; at least 0.0050%; at least 0.0100%; at least 0.020%; at least 0.100% at least 0.200%; at least 0.250%; at least 0.500% by weight of the feed additive.

In a further aspect there is provided the use of at least one amidase enzyme capable of degrading ochratoxin, preferably ochratoxin A, in the manufacture of a foodstuff or feedstuff for reducing the level of mycotoxin contamination in the foodstuff or feedstuff.

Preferably, the amidase enzyme is also capable of degrading at least one further ochratoxin or ochratoxin derivative and/or at least one ergot alkaloid, more preferably, ochratoxin B.

Preferably, the at least one amidase enzyme is formulated as a food or feed additive. More preferably, the food or feed additive is the food or feed additive according to the present invention.

In a further aspect of the present invention there is provided a composition comprising an ochratoxin, preferably ochratoxin A, contaminated material and an amidase which degrades ochratoxin, preferably ochratoxin A as described herein.

In a preferred embodiment of the present invention, the composition comprises a recombinant cell capable of expressing the ochratoxin A degrading amidase.

It will be understood that said composition can comprise any suitable ochratoxin A contaminated material. In preferred embodiments the composition comprises a food or feed material, a fermentation broth or waste product of the fermentation process such as DDGS, waste water or contaminated soil.

The skilled person will understand that ochratoxin A contamination of compositions other than food and feed materials can be problematical. For example, waste water containing ochratoxin A from industrial processes can result in contamination of water ways.

Also provided by the present invention, is a method of reducing ochratoxin contamination in a material, comprising adding to the contaminated material an amidase enzyme capable of degrading ochratoxin, preferably ochratoxin A.

Also provided by the present invention is a food or feed material obtainable by the methods of the present invention.

In a further aspect of the present invention there is provided a method of increasing the growth rate and/or health of an animal comprising feeding the animal an effective amount of a feedstuff according to the present invention.

As used in the present context, the term health refers to a reduction in the detrimental effects on an animal caused by ochratoxin toxicity resulting from levels of ochratoxin present in the feedstuff.

EXAMPLES

The invention will be further described with reference to the examples and figures in which:—

FIG. 1 shows the ability of PEPAd (a Danisco aspartic acid protease cloned from A. niger), Amano™ lipase and bovine Carboxypeptidase A at the same protein concentrations to breakdown OTA. Activity was expressed in % OT-α (product of OTA) area: the area corresponding to the product divided by the total area (peak area of OTA plus OT-α).

FIG. 2 shows the percentage of OT-α area and relative lipase activity (%) in fractions from (A): hydrophobic interaction chromatography (HIC)/affinity chromatography with a Phenyl Sepharose packed column (2.6 cm×10 cm) and gradient from 1M ammonium sulfate to 0. (B): Anion exchange chromatography (AEX) 030 column with gradient from 0 to 1M NaCl after HIC.

FIG. 3 shows SDS-PAGE analysis of the concentrated AEX fraction having highest OTA degrading activity. The indicated bands A and B were in-gel digested with trypsin and the digested peptides were separated on reverse phase HPLC and analyzed with MS for peptide identification.

FIG. 4 shows graphically the separation of OTA active fractions by HIC/affinity chromatography after ammonium sulfate fractionation. FIG. 4A shows HIC or affinity chromatography on Phenyl Sepharose column; FIG. 4B shows HIC on Butyl Sepharose column. FIG. 4C shows the lipase and OTA activity distribution in the collected fractions 2-28 from the Butyl Sepharose column.

FIG. 5 shows SDS-PAGE analysis of concentrated OTA active fractions after the purification steps of ammonium fractionation, HIC/affinity on Phenyl Sepharose, HIC on Butyl Sepharose, AEX on Source Q30 and membrane separation/concentration on Amicon centriprep 10 concentrator (molecular cutoff of 10 kDa).

FIG. 6 shows the activities present in the membrane separated and concentrated AEX fractions of FIG. 5 on formamide (FIG. 6A) and OTA (FIG. 6B), protein peaks assayed at 280 nm.

FIG. 7 shows the construction of the amidase 2 expression cassette.

FIG. 8 shows SOS-PAGE analysis of intracellular proteins of A. niger transformants harboring the Amidase 2 gene. The protein band of amidase 2 is indicated by an arrow bar.

FIG. 9 shows SDS-PAGE analysis of extracellular proteins of A. niger transformants harboring the Amidase 2 gene. The protein band of amidase 2 is indicated by an arrow bar.

FIG. 10 shows the screening of intracellular and extracellular (fermentation broth) fractions of Aspergillus niger transformants (1-15) harboring the amidase 2 gene for their ability to break down OTA. 10 μl of the intracellular fractions or the broth was mixed with 25 μL 1 μg/ml OTA, 100 μl sodium phosphate (67 mM, pH7.0) and incubated at 40° C. for 35 min. At the end of the reaction 130 μl acetonitrile containing 0.2% acetic acid was added to stop the reaction. The reaction mixture was filtered and 5 μl analysed by RP-HPLC analysis of remaining OTA. wt refers to the parent strain used for the transformation; ctrl, refers to the reaction in the absence of A. niger transformants.

FIG. 11 shows the pH optimum of the amidase 2 from transgenic A. niger intracellular preparation in degrading OTA. The reaction mixture contained 5 μl amidase 2 mixture (a mixture of the intracellular fractions of transformant 3, 4, 7-8, 10-11 and 13-15) with a protein concentration of 155 μg protein/ml), 0.1 ml 67 mM phosphate buffer (pH3, 4, 5, 6, 7, 8 and 9), 25 μl OTA (1 μg/ml). The reaction was performed at 30° C. for 60 min and stopped by the addition of 0.13 ml acetonitrile containing 0.2% HAC. An injection volume of 10 μl was used for RP-HPLC analysis of remaining OTA after filtering.

FIG. 12 shows the heat stability of amidase 2 from transgenic A. niger intracellular preparation. The pre-incubation mixture in a 1.5 ml ependorpf tube contained 5 μl of the amidase 2 mixture (155 μg protein/ml), and 45 μl 67 mM phosphate buffer pH7.0. The samples were pre-incubated at 80° C. for 0, 5, 10, 15, 20, 25 and 30 min. After cooling to 15° C., 50 μl 67 mM phosphate buffer and 25 μl OTA (1 μg/ml) were added to start the reaction this was incubated at 30° C. for 60 min, the reaction was stopped by the addition of 130 μl acetonitrile containing 0.2% HAC. An injection volume of 10 μl was used for RP-HPLC analysis of remaining OTA after filtering.

FIG. 13 shows pH and temperature stability of amidase 2 from transgenic A. niger inrtracellular preparation at incubated at 70° C. at pH 3-9 in 67 mM sodium phosphate. The reaction mixture contained 5 μl amidase 2 mixture (155 μg protein/ml), 45 μl 67 mM phosphate buffer at either pH3, 4, 5, 6, 7, 8 or 9. The reaction mixes were incubated at 70° C. for 30 min, before 2 μl 0.5 mg/ml OTA in ethanol and 80 μl 0.2M Mops pH 7.0 were added and the samples incubated at 30° C. for 60 min. The reaction was stopped by adding 130 μl acetonitrile containing 0.2% HAC, filtered and analysed by RP-HPLC.

FIG. 14 shows analysis of recombinant amidase 2 purified from the broth of A. niger transformant 13 by SDS-PAGE on AEX fractions (A) and by mass-spectrometry (B) on fraction 13 after AEX.

FIG. 15 shows OTA degradation as a function of amidase protein concentration. The amidase used was purified from the broth of transformant 13 (438aa protein referred to as amidase 2 mat).

FIG. 16 shows the effect of calcium ion concentration on the activity of amidase 2 mat purified from the transgenic A. niger and the Amano™ lipase product.

FIG. 17. Degradation of OTA by the amidase 2 extracted from the intracellular fraction (Amidase 2 sig) of A. niger. No inhibition by CaCl₂ or EDTA to amidase 2 sig is seen.

FIG. 18A shows the amidase 2 amino acid sequence (480aa) (SEQ ID NO: 1) from Aspergillus niger in one letter format. Single underlined sequences are those obtained from FIG. 5; double underlined are those obtained from FIG. 8.

FIG. 18B shows the amidase 2 amino acid sequence (480aa) plus 6 histidine residues at its C-terminal from Aspergillus niger in one letter format (SEQ ID NO. 4).

FIG. 19 shows the amidase 2 amino acid sequence (480aa) (SEQ ID NO: 1) from Aspergillus niger in three letter format. Features characteristic of OTA-degrading amidases are indicated as bold and underlined amino acid residues: conserved residues (bold) and nine sequence motifs.

FIG. 20 a shows the structure of the amidase 2 (SEQ ID NO:1) protein tetramer forming the distorted TIM-like barrel structure; b shows the structure of 1 of the 8 subunits.

FIG. 21 shows the structure of the amidase 2 active site environment.

FIG. 22 shows the crystal co-ordinates of a monomer of amidase 2 (SEQ ID NO: 1).

FIG. 23 shows a schematic representation of the expression plasmid pTTT-pyrG13-Amidase G. graminicola.

FIG. 24 shows a schematic representation of the expression plasmid pTTT-pyrG13-Amidase M. anisopliae.

FIG. 25 shows a map of the expression plasmid pRAX-Amidase A. oryzae.

FIG. 26 shows an alignment of 11 amidase sequences (SEQ ID NO: 1, SEQ ID NO: 5-11, and SEQ ID NO: 13-15) showing the 9 sequence motifs that are essential for amidase activity degrading ochratoxin.

FIG. 27 shows an alignment of SEQ ID NO:1 and carboxypeptidases A (SEQ ID NO: 16) and Y (SEQ ID NO: 17) which are known to have low ochratoxin degrading activity.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Singleton, at al., DICTIONARY OF MICROBIOLOGY AND MOLECULAR BIOLOGY, 20 ED., John Wiley and Sons, New York (1994), and Hale & Marham, THE HARPER COLLINS DICTIONARY OF BIOLOGY, Harper Perennial, NY (1991) provide one of skill with a general dictionary of many of the terms used in this disclosure.

This disclosure is not limited by the exemplary methods and materials disclosed herein, and any methods and materials similar or equivalent to those described herein can be used in the practice or testing of embodiments of this disclosure. Numeric ranges are inclusive of the numbers defining the range. Unless otherwise indicated, nucleic acid sequences are written left to right in 5′ to 3′ orientation; amino acid sequences are written left to right in amino to carboxy orientation, respectively.

The headings provided herein are not limitations of the various aspects or embodiments of this disclosure which can be had by reference to the specification as a whole. Accordingly, the terms defined immediately below are more fully defined by reference to the specification as a whole.

Amino acids are referred to herein using the name of the amino acid, the three letter abbreviation or the single letter abbreviation.

As used herein, the term “identity” means an entity having a certain homology with the amino acid sequences and the nucleotide sequences. The term “identity” in this context refers to the percentage of sequence identity between two enzymes after aligning their sequences using alignment algorithms as described in more detail below.

In the present context, a homologous amino acid sequence is taken to include an amino acid sequence which may be at least 30, 40, 50, 60, 70, 75, 80, 85 or 90% identical, preferably at least 95, 96, 97, 98 or 99% identical to the sequence. Typically, the homologues will comprise the same active sites etc.—e.g. as the subject amino acid sequence. Although homology can also be considered in terms of similarity (i.e. amino acid residues having similar chemical properties/functions A), in the context of the present invention it is preferred to express homology in terms of sequence identity.

For amino acid sequences and nucleotide sequences, homology comparisons can be conducted by eye, or more usually, with the aid of readily available sequence comparison programs. These commercially available computer programs can calculate % homology between two or more sequences.

% homology may be calculated over contiguous sequences, i.e. one sequence is aligned with the other sequence and each amino acid in one sequence is directly compared with the corresponding amino acid in the other sequence, one residue at a time. This is called an “ungapped” alignment. Typically, such ungapped alignments are performed only over a relatively short number of residues.

Although this is a very simple and consistent method, it fails to take into consideration that, for example, in an otherwise identical pair of sequences, one insertion or deletion will cause the following amino acid residues to be put out of alignment, thus potentially resulting in a large reduction in % homology when a global alignment is performed. Consequently, most sequence comparison methods are designed to produce optimal alignments that take into consideration possible insertions and deletions without penalising unduly the overall homology score. This is achieved by inserting “gaps” in the sequence alignment to try to maximise local homology.

However, these more complex methods assign “gap penalties” to each gap that occurs in the alignment so that, for the same number of identical amino acids, a sequence alignment with as few gaps as possible—reflecting higher relatedness between the two compared sequences—will achieve a higher score than one with many gaps. “Affine gap costs” are typically used that charge a relatively high cost for the existence of a gap and a smaller penalty for each subsequent residue in the gap. This is the most commonly used gap scoring system. High gap penalties will of course produce optimised alignments with fewer gaps. Most alignment programs allow the gap penalties to be modified. However, it is preferred to use the default values when using such software for sequence comparisons. For example when using the GCG Wisconsin Bestfit package the default gap penalty for amino acid sequences is −12 for a gap and −4 for each extension.

Calculation of maximum % homology therefore firstly requires the production of an optimal alignment, taking into consideration gap penalties. A suitable computer program for carrying out such an alignment is the GCG Wisconsin Bestfit package (Devereux et a/1984 Nuc. Acids Research 12 p 387). Examples of other software than can perform sequence comparisons include, but are not limited to, the BLAST package (see Ausubel et al., 1999 Short Protocols in Molecular Biology, 4^(th) Ed—Chapter 18), FASTA (Altschul et al., 1990 J. Mol. Biol. 403-410) and the GENEWORKS suite of comparison tools. Both BLAST and FASTA are available for offline and online searching (see Ausubel et al., 1999, Short Protocols in Molecular Biology, pages 7-58 to 7-60).

However, for some applications, it is preferred to use the GCG Bestfit program. A new tool, called BLAST 2 Sequences is also available for comparing protein and nucleotide sequence (see FEMS Microbiol Lett 1999 174(2): 247-50; FEMS Microbiol Lett 1999 177(1): 187-8 and tatiana@ncbi.nlm.nih.gov).

Although the final % homology can be measured in terms of identity, the alignment process itself is typically not based on an all-or-nothing pair comparison. Instead, a scaled similarity score matrix is generally used that assigns scores to each pairwise comparison based on chemical similarity or evolutionary distance. An example of such a matrix commonly used is the BLOSUM62 matrix—the default matrix for the BLAST suite of programs. GCG Wisconsin programs generally use either the public default values or a custom symbol comparison table if supplied (see user manual for further details). For some applications, it is preferred to use the public default values for the GCG package, or in the case of other software, the default matrix, such as BLOSUM62.

Alternatively, percentage homologies may be calculated using the multiple alignment feature in DNASIS™ (Hitachi Software), based on an algorithm, analogous to CLUSTAL (Higgins D G & Sharp P M (1988), Gene 73(1), 237-244).

Once the software has produced an optimal alignment, it is possible to calculate % homology, preferably % sequence identity. The software typically does this as part of the sequence comparison and generates a numerical result.

In a preferable aspect of the present invention the following software and settings for calculating percentage sequence homology/identity are used. For amino acid sequences percentage of identities (homology) or “positives” are calculated by the AlignX Vector NTI (Vector NTI Advance 9.1 from Invitrogen Corporation, Carlsbad, Calif., USA.) for each possible pair of amino acid sequences. Settings are default parameters (Gap opening penalty—10, Gap extension penalty 0.1).

For nucleic acid sequences percentage of identities (homology) or “positives” are calculated by the AlignX Vector NTI programme from informax Inc. (USA) for each possible pair of nucleic acid sequences. Settings are default settings for DNA are: Gap opening penalty: 15 and Gap extension penalty: 6.66 (same settings for multiple alignments).

Preferably the amino acid identity (homology) is calculated across the full-length amino acid sequence or for nucleic acid to a corresponding polynucleotide which encodes the respective the full-length amino acid sequence.

The sequences may also have deletions, insertions or substitutions of amino acid residues which produce a silent change and result in a functionally equivalent substance. Deliberate amino acid substitutions may be made on the basis of similarity in amino acid properties (such as polarity, charge, solubility, hydrophobicity, hydrophilicity, and/or the amphipathic nature of the residues) and it is therefore useful to group amino acids together in functional groups. Amino acids can be grouped together based on the properties of their side chain alone. However it is more useful to include mutation data as well. The sets of amino acids thus derived are likely to be conserved for structural reasons. These sets can be described in the form of a Venn diagram (Livingstone C. D. and Barton G. J. (1993) “Protein sequence alignments: a strategy for the hierarchical analysis of residue conservation” Comput. Appl Biosci. 9: 745-756)(Taylor W. R. (1986) “The classification of amino acid conservation” J. Theor. Biol. 119; 205-218). Conservative substitutions may be made, for example according to the table below which describes a generally accepted Venn diagram grouping of amino acids.

SET SUB-SET Hydro- F W Y H K M I L V A G C Aromatic F W Y H phobic Ali- I L V phatic Polar W Y H K R E D C S T N Q Charged H K R E D Posi- H K R tively charged Nega- E D tively charged Small V C A G S P T N D Tiny A G S

The present invention also encompasses homologous substitution (substitution and replacement are both used herein to mean the interchange of an existing amino acid residue, with an alternative residue) that may occur i.e. like-for-like substitution such as basic for

basic, acidic for acidic, polar for polar etc. Non-homologous substitution may also occur i.e. from one class of residue to another or alternatively involving the inclusion of unnatural amino acids such as ornithine (hereinafter referred to as Z), diaminobutyric acid ornithine (hereinafter referred to as B), norleucine ornithine (hereinafter referred to as O), pyriylalanine, thienylalanine, naphthylalanine and phenylglycine.

Replacements may also be made by unnatural amino acids.

The term “protein”, as used herein, includes proteins, polypeptides, and peptides.

The terms “amino acid residue equivalent to”, “amino acid corresponding to” and grammatical equivalents thereof are used herein to refer to an amino acid residue of a protein having the similar position and effect as that indicated in a particular amino acid sequence of a particular protein. The person of skill in the art will recognize the equivalence of specified residues in comparable proteins.

The term “property” or grammatical equivalents thereof in the context of a polypeptide, as used herein, refer to any characteristic or attribute of a polypeptide that can be selected or detected. These properties include, but are not limited to oxidative stability, substrate specificity, catalytic activity, thermal stability, temperature and/or pH activity profile, feed processing stability, and ability to be secreted.

As used herein, the term “amino acid sequence” is synonymous with the term “polypeptide” and/or the term “protein”. In some instances, the term “amino acid sequence” is synonymous with the term “peptide”. In some instances, the term “amino acid sequence” is synonymous with the term “enzyme”.

The amino acid sequence may be prepared/isolated from a suitable source, or it may be made synthetically or it may be prepared by use of recombinant DNA techniques.

The terms “protein” and “polypeptide” are used interchangeably herein. In the present disclosure and claims, the conventional one-letter and three-letter codes for amino acid residues are used. The 3-letter code for amino acids as defined in conformity with the IUPACIUB Joint Commission on Biochemical Nomenclature (JCBN). It is also understood that a polypeptide may be coded for by more than one nucleotide sequence due to the degeneracy of the genetic code.

The term “signal sequence” or “signal peptide” refers to any sequence of nucleotides and/or amino acids which may participate in the secretion of the mature or precursor forms of the protein. This definition of signal sequence is a functional one, meant to include all those amino acid sequences encoded by the N-terminal portion of the protein gene, which participate in the effectuation of the secretion of protein. They are often, but not universally, bound to the N-terminal portion of a protein or to the N-terminal portion of a precursor protein.

By “functional fragment” is meant a fragment of the polypeptide that retains that characteristic properties of that polypeptide. In the context of the present invention, a functional fragment of an amidase enzyme is a fragment that retains the amidase enzyme cleavage capability of the whole protein.

The term “isolated”, “recovered” or “purified” refers to a material that is removed from its original environment. The term “substantially purified” means that the material has been purified to at least a substantial degree.

In one aspect, preferably the nucleotide or amino acid sequence is in an isolated form. The term “isolated” means that the sequence is at least substantially free from at least one other component with which the sequence is naturally associated in nature and as found in nature.

Other definitions of terms may appear throughout the specification. Before the exemplary embodiments are described in more detail, it is to understand that this disclosure is not limited to particular embodiments described, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present disclosure will be limited only by the appended claims.

Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limits of that range is also specifically disclosed. Each smaller range between any stated value or intervening value in a stated range and any other stated or intervening value in that stated range is encompassed within this disclosure. The upper and lower limits of these smaller ranges may independently be included or excluded in the range, and each range where either, neither or both limits are included in the smaller ranges is also encompassed within this disclosure, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in this disclosure.

It must be noted that as used herein and in the appended claims, the singular forms “a”, “an”, and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a gene” includes a plurality of such candidate agents and reference to “the cell” includes reference to one or more cells and equivalents thereof known to those skilled in the art, and so forth.

The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that such publications constitute prior art to the claims appended hereto.

The enzymes for use in the present invention can be produced either by solid or submerged culture, including batch, fed-batch and continuous-flow processes. Culturing is accomplished in a growth medium comprising an aqueous mineral salts medium, organic growth factors, the carbon and energy source material, molecular oxygen, and, of course, a starting inoculum of one or more particular microorganism species to be employed.

In addition to the carbon and energy source, oxygen, assimilable nitrogen, and an inoculum of the microorganism, it is necessary to supply suitable amounts in proper proportions of mineral nutrients to assure proper microorganism growth, maximize the assimilation of the carbon and energy source by the cells in the microbial conversion process, and achieve maximum cellular yields with maximum cell density in the fermentation media.

The composition of the aqueous mineral medium can vary over a wide range, depending in part on the microorganism and substrate employed, as is known in the art. The mineral media should include, in addition to nitrogen, suitable amounts of phosphorus, magnesium, calcium, potassium, sulphur, and sodium, in suitable soluble assimilable ionic and combined forms, and also present preferably should be certain trace elements such as copper, manganese, molybdenum, zinc, iron, boron, and iodine, and others, again in suitable soluble assimilable form, all as known in the art.

The fermentation reaction is an aerobic process in which the molecular oxygen needed is supplied by a molecular oxygen-containing gas such as air, oxygen-enriched air, or even substantially pure molecular oxygen, provided to maintain the contents of the fermentation vessel with a suitable oxygen partial pressure effective in assisting the microorganism species to grow in a thriving fashion. In effect, by using an oxygenated hydrocarbon substrate, the oxygen requirement for growth of the microorganism is reduced. Nevertheless, molecular oxygen must be supplied for growth, since the assimilation of the substrate and corresponding growth of the microorganisms, is, in part, a combustion process.

Although the aeration rate can vary over a considerable range, aeration generally is conducted at a rate which is in the range of about 0.5 to 10, preferably about 0.5 to 7, ˜volumes (at the pressure employed and at 25° C.) of oxygen-containing gas per liquid volume in the fermentor per minute. This amount is based on air of normal oxygen content being supplied to the reactor, and in terms of pure oxygen the respective ranges would be about 0.1 to 1.7, or preferably about 0.1 to 1.3, volumes (at the pressure employed and at 25° C.) of oxygen per liquid volume in the fermentor per minute.

The pressure employed for the microbial conversion process can range widely. Pressures generally are within the range of about 0 to 50 psig, presently preferably about 0 to 30 psig, more preferably at least slightly over atmospheric pressure, as a balance of equipment and operating cost versus oxygen solubility achieved. Greater than atmospheric pressures are advantageous in that such pressures do tend to increase a dissolved oxygen concentration in the aqueous ferment, which in turn can help increase cellular growth rates. At the same time this is balanced by the fact that high atmospheric pressures do increase equipment and operating costs.

The fermentation temperature can vary somewhat, but for filamentous fungi such as Aspergillus niger or Trichoderma reesei the temperature generally will be within the range of about 20° C. to 40° C., generally preferably in the range of about 25° C. to 34° C., depending on the strain of microorganism chosen.

The microorganisms also require a source of assimilable nitrogen. The source of assimilable nitrogen can be any nitrogen-containing compound or compounds capable of releasing nitrogen in a form suitable for metabolic utilization by the microorganism. While a variety of organic nitrogen source compounds, such as protein hydrolysates, can be employed, usually cheap nitrogen-containing compounds such as ammonia, ammonium hydroxide, urea, and various ammonium salts such as ammonium phosphate, ammonium sulfate, ammonium pyrophosphate, ammonium chloride, or various other ammonium compounds can be utilized. Ammonia gas itself is convenient for large scale operations, and can be employed by bubbling through the aqueous ferment (fermentation medium) in suitable amounts. At the same time, such ammonia can also be employed to assist in pH control.

The pH range in the aqueous microbial ferment (fermentation admixture) should be in the exemplary range of about 2.0 to 8.0. With filamentous fungi, the pH normally is within the range of about 2.5 to 8.0; with Aspergillius niger or Trichoderma reesei, the pH normally is within the range of about 3.0 to 7.0. pH range preferences for certain microorganisms are dependent on the media employed to some extent, as well as the particular microorganism, and thus change somewhat with change in media as can be readily determined by those skilled in the art.

While the average retention time of the fermentation admixture in the fermentor can vary considerably, depending in part on the fermentation temperature and culture employed, generally it will be within the range of about 24 to 500 hours, preferably presently about 24 to 400 hours. Preferably, the fermentation is conducted in such a manner that the carbon-containing substrate can be controlled as a limiting factor, thereby providing good conversion of the carbon-containing substrate to cells and avoiding contamination of the cells with a substantial amount of unconverted substrate. The latter is not a problem with water-soluble substrates, since any remaining traces are readily washed off. It may be a problem, however, in the case of non-water-soluble substrates, and require added product-treatment steps such as suitable washing steps. As described above, the time to reach this level is not critical and may vary with the particular microorganism and fermentation process being conducted. However, it is well known in the art how to determine the carbon source concentration in the fermentation medium and whether or not the desired level of carbon source has been achieved.

Although the fermentation can be conducted as a batch or continuous operation, fed batch operation is much to be preferred for ease of control, production of uniform quantities of products, and most economical uses of all equipment. If desired, part or all of the carbon and energy source material and/or part of the assimilable nitrogen source such as ammonia can be added to the aqueous mineral medium prior to feeding the aqueous mineral medium to the fermentor. Each of the streams introduced into the reactor preferably is controlled at a predetermined rate, or in response to a need determinable by monitoring such as concentration of the carbon and energy substrate, pH, dissolved oxygen, oxygen or carbon dioxide in the off-gases from the fermentor, cell density measurable by light transmittancy, or the like. The feed rates of the various materials can be varied so as to obtain as rapid a cell growth rate as possible, consistent with efficient utilization of the carbon and energy source, to obtain as high a yield of microorganism cells relative to substrate charge as possible.

In either a batch, or the preferred fed batch operation, all equipment, reactor, or fermentation means, vessel or container, piping, attendant circulating or cooling devices, and the like, are initially sterilized, usually by employing steam such as at about 121° C. for at least about 15 minutes. The sterilized reactor then is inoculated with a culture of the selected microorganism in the presence of all the required nutrients, including oxygen, and the carbon-containing substrate. The type of fermentor employed is not critical, though presently preferred is operation under 15 L Biolafitte (Saint-Germain-en-Laye, France).

The collection and purification of the enzymes of the present invention from the fermentation broth can also be done by procedures known per se in the art. The fermentation broth will generally contain cellular debris, including cells, various suspended solids and other biomass contaminants, as well as the desired enzyme product of the present invention, which are preferably removed from the fermentation broth by means known in the art. Suitable processes for such removal include conventional solid-liquid separation techniques such as, e.g., centrifugation, filtration, dialysis, microfiltration, rotary vacuum filtration, or other known processes, to produce a cell-free filtrate. It may be preferable to further concentrate the fermentation broth or the cell-free filtrate using techniques such as ultrafiltration, evaporation or precipitation. Precipitating the proteinaceous components of the supernatant or filtrate may be accomplished by means of a salt, e.g., ammonium sulfate. Further purification may optionally be achieved by crystallization or by a variety of chromatographic procedures, e.g., ion exchange chromatography, affinity chromatography or similar art recognized procedures.

Variants/Derivatives

The present invention also encompasses the use of variants, homologues and derivatives of any amino acid sequence of an enzyme or of any nucleotide sequence encoding such an enzyme.

Variant amino acid sequences may include suitable spacer groups that may be inserted between any two amino acid residues of the sequence including alkyl groups such as methyl, ethyl or propyl groups in addition to amino acid spacers such as glycine or β-alanine residues. A further form of variation, involves the presence of one or more amino acid residues in peptoid form, will be well understood by those skilled in the art. For the avoidance of doubt, “the peptoid form” is used to refer to variant amino acid residues wherein the α-carbon substituent group is on the residue's nitrogen atom rather than the α-carbon. Processes for preparing peptides in the peptoid form are known in the art, for example Simon R J at al., PNAS (1992) 89(20), 9367-9371 and Norwell D C, Trends Biotechnol. (1995) 13(4), 132-134.

Other Components

The feed additives or compositions of the present invention may be used in combination with other components or carriers.

Suitable carriers for feed enzymes include maltodextrin, limestone (calcium carbonate), cyclodextrin, wheat, wheat bran or a wheat component, rice or rice bran, sucrose, starch, Na₂SO₄, Talc, PVA and mixtures thereof. In addition there are a number of encapsulation techniques including those based on fat/wax coverage, adding plant gums etc.

Examples of other components include one or more of: thickeners, gelling agents, emulsifiers, binders, crystal modifiers, sweeteners (including artificial sweeteners), rheology modifiers, stabilisers, anti-oxidants, dyes, enzymes, carriers, vehicles, excipients, diluents, lubricating agents, flavouring agents, colouring matter, suspending agents, disintegrants, granulation binders etc. These other components may be natural. These other components may be prepared by use of chemical and/or enzymatic techniques.

As used herein the term “thickener or gelling agent” as used herein refers to a product that prevents separation by slowing or preventing the movement of particles, either droplets of immiscible liquids, air or insoluble solids.

The term “stabiliser” as used here is defined as an ingredient or combination of ingredients that keeps a product (e.g. a food product) from changing over time.

The term “emulsifier” as used herein refers to an ingredient (e.g. a food product ingredient) that prevents the separation of emulsions.

As used herein the term “binder” refers to an ingredient (e.g. a food ingredient) that binds the product together through a physical or chemical reaction.

The term “crystal modifier” as used herein refers to an ingredient (e.g. a food ingredient) that affects the crystallisation of either fat or water.

“Carriers” or “vehicles” mean materials suitable for compound administration and include any such material known in the art such as, for example, any liquid, gel, solvent, liquid diluent, solubiliser, or the like, which is non-toxic and which does not interact with any components of the composition in a deleterious manner.

Examples of nutritionally acceptable carriers include, for example, grain, water, salt solutions, alcohol, silicone, waxes, petroleum jelly, vegetable oils, and the like.

Examples of excipients include one or more of: microcrystalline cellulose and other celluloses, lactose, sodium citrate, calcium carbonate, dibasic calcium phosphate, glycine, starch, milk sugar and high molecular weight polyethylene glycols.

Examples of disintegrants include one or more of: starch (preferably corn, potato or tapioca starch), sodium starch glycollate, croscarmellose sodium and certain complex silicates.

Examples of granulation binders include one or more of: polyvinylpyrrolidone, hydroxypropylmethylcellulose (HPMC), hydroxypropylcellulose (HPC), sucrose, maltose, gelatin and acacia.

Examples of lubricating agents include one or more of: magnesium stearate, stearic acid, glyceryl behenate and talc.

Examples of diluents include one or more of: water, ethanol, propylene glycol and glycerin, and combinations thereof.

The other components may be used simultaneously (e.g. when they are in admixture together or even when they are delivered by different routes) or sequentially (e.g. they may be delivered by different routes).

As used herein the term “component suitable for animal or human consumption” means a compound which is or can be added to the composition of the present invention as a additive which may be of nutritional benefit, a fibre substitute or have a generally beneficial effect to the consumer.

By way of example, the components may be prebiotics such as alginate, xanthan, pectin, locust bean gum (LBG), inulin, guar gum, galacto-oligosaccharide (GOS), fructo-oligosaccharide (FOS), lactosucrose, soybean oligosaccharides, palatinose, isomalto-oligosaccharides, gluco-oligosaccharides and xylo-oligosaccharides.

Isolated

In one aspect, preferably the amidase enzyme for use in the present invention is in an isolated form. The term “isolated” means that the amidase enzyme is at least substantially free from at least one other component with which the amidase enzyme is naturally associated in nature and as found in nature. The term “isolated” may mean that the amidase enzyme is at least substantially free from at least one other component in the culture media in which it is produced. The amidase enzyme of the present invention may be provided in a form that is substantially free of one or more contaminants with which the substance might otherwise be associated or with which the enzyme may be produced.

Thus, for example it may be substantially free of the cell(s) or one or more potentially contaminating polypeptides and/or nucleic acid molecules. The amidase enzyme may be isolated by separating the cell(s) from the broth during or after fermentation so that the amidase enzyme remains in the broth. The amidase enzyme may be isolated by subjecting the fermentation broth to cell separation by vacuum filtration.

In one embodiment the term isolated means that the amidase is isolated from the broth such that it substantially free from other components in the culture media in which it is produced.

Purified

In one aspect, preferably the amidase enzyme for use in the present invention is in a purified form. The term “purified” means that the given component is present at a high level. The component is desirably the predominant component present in a composition. Preferably, it is present at a level of at least about 30%, 40%, 50%, 60%, or at least about 65%, or at least about 70%, or at least about 75%, or at least about 80%, or at least about 85%, or at least about 90%, or at least about 95%, or at least about 97%, or at least about 99% said level being determined on a dry weight/dry weight basis with respect to the total composition under consideration. For some embodiments the amount is at least about 85% said level being determined on a dry weight/dry weight basis with respect to the total composition under consideration.

Concentrate

In one aspect, preferably the amidase enzyme for use in the present invention is used as a concentrate. The concentrate may be a concentrated form of the medium into which the enzyme has been excreted. Preferably, the concentrate may be a concentrated form of the medium into which the enzyme has been secreted and wherein the cell(s) have been removed.

Materials and Methods

Ochratoxin A was from Fluka (cat. no. 32937-5MG). Carboxypeptidase A from bovine pancreas (Sigma, cat. No. 09268). All other enzymes including commercial available amidases and reagents were from Sigma, Roche, Merck and Danisco. Amano™ lipase was from Amano Enzyme Inc. (Nagoya, Japan). All chromatgraphy medium 1.0 including Phenyl-, Octyl-Butyl-Sepharose, anion Exchange, gel filtration (PD10 column) and protein purification apparatus Äkta explorer were from GE Healthcare. Microplates were from Nunc A/S (Denmark) and Corning (USA). Microplate reader was from Biotek (USA). Corn flour was obtained from local market while soy-corn based feed was obtained from The Danish Technological Institute at Kolding (Denmark).

Mycotoxin Degradation Assays:

Method for Measuring Formamidase Activity

Solutions used: Megazyme formic acid assay kit (K-form 05/06) Bottle 1, add 4 ml milliQ water to produce potassium phosphate buffer pH7.6. Keep at 5° C.; Bottle 2, add 5.2 ml water to obtain the right NAD concentration (Kept at −20° C.); Bottle 3, is FDH shake before use (Kept it at 5° C.). Formamide is a liquid product at 99.80% from Sigma (F9037) (kept at 5° C.) and was used as it was without further dilution.

Assay procedure: to 96 well half area uv transparent microplate add:

-   -   10 μl potassium phosphate buffer (pH 7.6) (Solution 1 from the         kit),     -   10 μl NAD (Solution 2 from the kit),     -   2.5 μl formic acid dehydrogenase (FDH)(Solution 3 from the kit),     -   1.6 μl 100% (v/v) formamide solution (from Sigma, F9037),     -   100 μl milliQ water.

At 37° C. in a microplate reader, read OD increase at 340 nm every min and shake before each reading for 15-60 min. Reading interval: 1 min.

After 5 min reading, add 5 μl-20 μl A. niger preparation (depending on the activity). Get the reaction rate (maximum linear slope).

Control: no formamidase was added but add buffer or water or cell-extract having no formamidase activity instead.

Determination of Enzymatic Degradation of OTA.

The buffers employed in the assay were: 50 mM sodium acetate buffer (pH 4, 4.5, 5 and 5.5); 50 mM MES buffer (pH 6, 6.5 and 7); 0.2M Mops-NaOH) pH7.0), 50 mM Tris-HCl buffer (pH 7.5, 8 and 8.5). Ochratoxin A (OTA) solution (1 mg/mL) was made of 5 mg ochratoxin A (Sigma, ref. 32937) dissolved in ethanol/water (60/40) and stored at −20° C. Screening assays were performed with 154 OTA solution and different enzyme concentrations (for example, 0.1-5 mg/ml) in a final volume of 300 μL. Enzymatic reactions were done at 37° C. for 20 h±1 h under constant agitation. Samples were filtered using syringe filter 0.20 μm for HPLC analysis. The HPLC apparatus consisted of a Dionex P580 pump and Dionex ASI-100 autosampler connected to a Dionex RF-2000 fluorescence detector (λ_(ex)=333 nm; λ_(em)=460 nm) and a Dionex UVD340 U UV detector at 257 nm. Elution was through a nucleosil 100-5 C18 column (250×4.6 mm, 5 μm particle size; Chrompack) with water/acetonitrile/acetic acid (100:100:1, v/v/v) at a 0.6 mL/min flow-rate. The column was kept at 30° C. and was connected to guard column (C18, 1 mm, Optimize technologies). The OTA and its degradation products were monitored both by uv at 235 nm and fluorescence (excitation at 278 nm emission at 440 nm).

Assay of A. niger Preparations for OTA Activity.

The reactions mixture consisted of 245 μL OTA (1 μg/mL) diluted in buffer pH7.0 of 50 mM Mes-NaOH and 54. of sample (A. niger broth, cell-free extract, fractions after ammonium fractionation and chromatography or product from A. niger). The reaction was performed at 40° C. for 30 min to 2 hours. Heating at 95° C. for 5 min was used to stop the reaction. Samples were filtered using syringe filter 0.20 μm and 54 injected in the HPLC apparatus.

Lipase and Esterase Assay.

Lipase and esterase activity were assayed using p-nitrophenyl butyrate (pNPB) purchased from Sigma as the substrate. Five microliters pNPB 40 mM dissolved in acetonitrile was added to 165 μL of 50 mM Tris-HCl buffer pH 7.5 and 5 μL of enzyme solution and used to test OTA degradation. Optical density was followed at 410 nm for 60 min. Protease Activity Assay (Petri Dish Method).

Protease activity was confirmed using plates containing 1% casein, 1% agarose in 100 mM di-sodium hydrogen phosphate buffer pH 6. The plate was incubated overnight at 37° C. and the protease activity was revealed by the development of a whitish ring around the well containing enzyme.

Protein Concentration Assay.

Protein concentration was determined with Bio-rad protein assay reagent based on the method of Bradford according to the procedure provided by the manufacturer. Using BSA as standard, the OD was measured in microplate at 595 nm, each samples dosed in duplicate.

Purification of OTA Degrading Activity from A. niger

Sample Preparation: Ammonium Sulfate Fractionation.

Five grams of Amano lipase enzyme were dissolved in 100 mL of 50 mM Tris/HCl buffer pH 7.5. The amount of ammonium sulfate corresponding to 40% saturation was added and crystals dissolved. After 10 min centrifugation at 3500 rpm, the supernatant was collected and ammonium sulfate added to reach 60% saturation. Crystals were dissolved by stirring and the solution centrifuged again. The pellet corresponding to the precipitate from 40% to 60% ammonium sulfate saturation was dissolved in 4×40 mL of 50 mM tricine, 1M ammonium sulfate pH7 and filtered with 0.22 μm filter.

Hydrophobic Interaction Chromatography (HIC).

The sample from ammonium sulfate fractionation was injected and applied to either Phenyl Sepharose FF, Butyl- or Octyl-Sepharose CL4B column equilibrated with 50 mM tricine containing 1M ammonium sulfate pH 7 (buffer A). The column, connected to Akta purifier system, was washed with buffer A (10 mL/min) and the bound proteins were eluted with a linear gradient of 50 mM tricine (Buffer B). Fractions of 10 mL were collected and used for OTA activity assay or formamidase assay.

Anion Exchange Chromatography (AEX).

Fractions with high conversion of OTA were pooled and desalted on a PD10 column equilibrated in 20 mM Tris-HCl, pH 7.5 (buffer A). The desalted sample was applied to a Source Q30 column equilibrated in buffer A (10 ml/min). The column was washed with buffer A and the bound proteins were eluted with a linear gradient of 0-1 M NaCl in buffer A (Buffer B is the same as Buffer A containing 1M NaCl). During the gradient, fractions of 5 ml were collected.

Concentration of Protein.

Fractions with OTA degrading activity obtained by anion exchange chromatography were concentrated using Amicon Centriprep™ concentrators with regenerated cellulose membrane (molecular cutoff 10 kDa).

Optimum pH Determination.

Five microliters of enzyme solution were combined with 2454 OTA 1 μg/mL diluted in different buffers: 50 mM sodium acetate buffer (pH 4, 4.5, 5 and 5.5); 50 mM MES-NaOH buffer (pH 6, 6.5 and 7); 50 mM Tris-HCl buffer (pH 7.5, 8 and 8.5). Reactions were performed at 37° C. for 30 min and stopped by heating at 95° C. for 5 min.

Optimum Temperature.

Five microliters of enzyme solution were mixed with 245 μL OTA 1 μg/mL at optimum pH and incubated at 20, 30, 40, 60, 70° C. for 30 min. Reactions were stopped by heating at 95° C. for 5 min.

Temperature Stability.

The enzyme solution was incubated at different temperature (30, 40, 60, 70, 80 and 90° C.) for 1 h and 5 μL were mixed with 2454 OTA 1 μg/mL for ochratoxin degradation reaction under optimum conditions. Reactions were stopped by heating at 95° C. for 5 min. The control was not incubated.

SDS-PAGE.

To separate and analyze the proteins sodium dodecyl sulfate polyacrylamide gel eletrophoresis, (SDS-PAGE) was used. Forty microliter samples were mixed with 10 μL 5× loading buffer (containing DDT and SDS), and boiled for 5 min. 20 μL were loaded on a NuPAGE 4-12% Bis-Tris gel (Invitrogen, USA). The electrophoresis was run according to the procedure from the manufacturer. The gel was stained with Coomassie brilliant blue.

Mass Spectrometry.

The protein of interest was cut from the gel (as above) using a scalpel and transferred to an Eppendorf tube. The protein was digested with an enzyme (primarily trypsin). After digestion peptides were extracted and analyzed by HPLC-MS/MS. The MS/MS spectra were automatically searched against protein databases for the purpose of idenity and characterisation.

Inhibition Assays for Formamidase and Amidase 2.

One protease inhibitor cocktail (PIC) tablet (Roche Applied Science, Mannhein, Germany) was dissolved in 1.5 mL. milliQ water to obtain a 7× stock solution. For formamidase inhibition assay, instead of 100 μL milliQ water normally employed, 20 μL of 7×PIC stock solution and 80 μL of milliQ water were mixed with other components listed in formamidase activity assay section. For OTA degrading activity, 35 μL 7×PIC solution were mixed with 210 μL OTA 1 μg/mL and 5 μL enzyme solution. A 125 mM EDTA stock solution was prepared and appropriate volumes to obtain 10 mM and 50 mM final concentration were mixed with OTA 1 μg/mL and 54 enzyme. For controls, inhibitor solution is replaced either by milliQ water for formamidase activity assay or MES-NaOH pH7 buffer for OTA degrading activity.

RESULTS Example 1 Screening for OTA Activity

Thirty enzymes including various lipases, proteases and amdiases were investigated for their ability to degrade ochratoxin A (Table 1). Only carboxypeptidase A and lipase from A. niger (Amano™ A) were able to breakdown OTA as previously reported (Pitout, 1969; Stander et al., 2000). Moreover, aspartic acid protease pepAd2 (Danisco) from A. niger showed an OTA converting activity although this was less efficient. To facilitate the comparison of these three enzymes, the protein concentration of PEPAd was determined and enzyme solutions of carboxypeptidase A and Amano™ lipase A were prepared at the same concentration (1 mg/ml). A kinetic of the degradation was carried out (FIG. 1). FIG. 1 shows that the OTA degradation kinetics of Amano™ lipase A and carboxypeptidase A were similar. After 7 h reaction at 37° C., no OTA peak was visible, meaning that full conversion of OTA was obtained. For PEPAd, full conversion was not reached even after 1 day reaction. Amano™ lipase A product was a mixture of different enzymes with the dominant activity being lipase. Besides PEPAd, other A. niger aspartic acid proteases of PEPAa, PEPAb and PEPAc (Wang, 2010), porcine elastase, and yeast carboxypeptiase Y all showed certain activity with OTA. This is the first time aspartic proteases other than metallo-protease (i.e., carboxypeptidase A, a Zn²⁺ dependent protease), serine protease elastase and carboxypeptidase Y have been shown to be able to break OTA down. Other proteases, lipases and amidases listed in Table 1 were not able to break OTA down (data not shown).

TABLE 1 Enzymes screened for ochratoxin A degradation Enzyme Organism Ref pH Manufacturer Carboxypeptidase A Bovine pancreas C9268 7.5 Sigma Carboxypeptidase Y Yeast 12758 7 USB Carboxypeptidase Y Yeast C3888 7 Sigma Chymotrypsin 8 Christian Chymostar Hansen Elastase Porcine prancreas E0258 8.5 Sigma Endoproteinase Glu-C S. Aureus 791156 7.5 Roche Diagnostics LG12 Bacillus subtilis 7.5 Danisco Lipase A Amano ™ Aspergillus niger 534781 7.5 Amano ™ PROACT #3617 Nocardiopsis prasina 7.5/4 Novozymes Proline peptidase proC 7 Protease Bacillus P1512 8 Sigma thermolysin thermoproteolyticus rokko Proteinase K Tritirachium album p0390 8 Sigma Protease P3000 Bacillus subtilis 7.5 Trypsin Bovine pancreas 109819 8 Roche Diagnostics phospholipase KLM1 7 Danisco lipase 3 5.5 Danisco Lipase Rhizopus arrhizus 62305 8.5 Sigma Lipase Candida cylindracea 62316 6.5 Sigma Lipase Rhizopus niveus 62310 7.5 Sigma GAP3 Aspergillus Niger 6 Danisco PEPAa Aspergillus Niger 6 Danisco PEPAb Aspergillus Niger 6 Danisco PEPAc Aspergillus Niger 6 Danisco PEPAd1 (c-terminal deletion) Aspergillus Niger 6 Danisco PEPAd2 (full length) Aspergillus Niger 6 Danisco Grindamyl Aspergillus Niger 7.5 Danisco Amidase Pseudomonas A6691 7 Sigma aeruginosa Peptide Amidase Citrus sinensis 17232 7 Fluka Amidase PLY511 7 Profors Penicillin amidase E. coli 76429 7 Sigma

Example 2 Purification of Ochratoxin A Degrading Activity from A. niger Fermentation Liquid or Filtered Broth (Cell Free)

Firstly ammonium sulfate precipitation was employed. Several ammonium sulfate concentrations were applied to an A. niger crude protein extract. The OTA degrading activity was determined in both supernatant and pellet. By increasing the ammonium sulfate concentration, the OTA degrading activity was transferred from the supernatant to the pellet. At 60% ammonium sulfate, all the degrading activity was found in the pellet. This demonstrated that the enzyme was able to hydrolyze the mycotoxin precipitates from 40% to 60% ammonium sulfate. At the same salt concentration (60%), a slight lipase activity remained in the pellet (data not shown).

The ammonium sulfate fraction from 40% to 60% was employed as the starting material for further ochratoxin degrading enzyme purification. Hydrophobic interaction chromatography (HIC) followed by anion exchange chromatography (AEX) as described above were used to separate lipase activity from OTA degrading activity. The active fraction was suspended in 50 mM tricine-HCl (pH7.0) containing 1M (NH₄)₂SO₄ (Buffer A).

FIG. 2A shows the percentage of OT-alpha area obtained after 1 h reaction of the fractions obtained from Phenyl Sepharose with OTA. This figure shows an increase in OTA degrading activity from fraction no. 51 to no. 64, while the highest lipase/esterase activity was found in fraction no. 65-68. This observation indicates that the ochratoxin degrading activity had apparently nothing to with the lipase/esterase activity in the Amano™ lipase product (it has previously been indicated that OTA degrading activity was due to lipase activity, Stander at al., 2000). The protein that degrades OTA was eluted between 90% and 100% buffer B (50 mM tricine-HCl (pH 7.0), indicating that Phenyl Sepharose chromatography medium apparently functioned as an affinity medium. OTA also contains a phenyl group and the OTA degrading enzyme must be able to bind the phenyl group from either OTA or the separation medium used here. Concerning the lipase/esterase activity, which was present in all fractions obtained from this step, it was eluted at 100% buffer B. This is not unexpected because lipases/esterases perform hydrolysis of lipids and triglycerides, which are very hydrophobic substrates and therefore bind tightly with hydrophobic matrices like Phenyl-Sepharose.

FIG. 2B shows the percentage of OT-alpha area obtained after 30 min reaction of the AEX fractions with OTA. The maximum OTA activity was found in fraction no. 26. However, it was also in these fractions that the relative lipase activity was the highest. This indicates that the AEX step was not able to separate lipase/esterase from OTA degrading activity, this was used by earlier investigators as the sole chromatography step in their attempt to purify OTA degrading activity (Abrunhosa at al., 2007).

In summary FIGS. 2A and 2B indicate that lipase/esterase is not the enzyme that is responsible for the degradation of OTA (FIG. 2A) as it has been assumed earlier (Stander et al. (2000) and the separation of lipase/esterase activity from OTA degrading activity was not achieved by AEX, which was practiced by earlier investigators in an effort to purify the enzyme degrading OTA from Amano™ lipase (Abrunhosa at al., 2007). The earlier investigators did not show that Phenyl Sepharose chromatography medium usually regarded as an hydrophobic interaction chromatography medium separating proteins based on their difference in hydrophobicity could be used here as an affinity separation medium for OTA degrading activity.

After the purification by ammonium sulfate fractionation followed by HIC/affinity chromatography on Phenyl Seharose and AEX, the fractions which contained the OTA degrading activity still had some lipase/esterase activity. FIG. 4 shows the results of analysis of the active fractions on OTA obtained in the AEX purification procedure of FIG. 2B by SDS-PAGE. It can be seen that on the gel there are two major bands: A and B. The partial sequencing of those bands by mass spectrometry followed by homology searching in the protein database suggests that Band A comprises at least 3 proteins corresponding respectively to:

1—Predicted acetamidase/formamidase from Aspergillus oryzae 45,012Da (27% coverage)

-   -   2—Formamidase from Aspergillus fumigatus 45,277Da (24% coverage)     -   3—Formamidase from Aspergillus nidulans 44,907Da (20% coverage)

And that Band B comprises at least 3 proteins corresponding to:

-   -   1—Glyoxylate/hydroxypyruvate reductase from Aspergillus oryzae         39,603 Da (34% coverage)     -   2—Acetylesterase from Aspergillus awamori 32,640 Da (9%         coverage)     -   3—Endo-1,4-beta-xylanase A precursor from Aspergillus niger         35,486 Da (17% coverage).

From the results indicated in FIG. 4, it appeared likely that the enzyme which breaks down OTA was A. niger formamidase. The other identified enzymes have theoretically nothing to do with OTA degradation. Further purification of A. niger formamidase was undertaken.

Example 3 Improved Purification of Ochratoxin A Degrading Enzyme

Further improvement for the purification by HIC and AEX was not achieved by using different buffers and different gradient programs. Purification using Phenyl Sepharose can be regarded as an affinity step because the OTA molecule has the phenyl group however, the impurities have high hydrophobicity and bind tightly to the phenyl Sepharose medium and therefore elute very late (FIG. 4A). When Octyl- and Butyl Sepharose were employed, the OTA degrading activity fraction was found in the unadsorbed flow-through fractions. This indicates that it has low hydrophobicity and that its tight biding to Phenyl Sepahrose is of affinity in nature (FIG. 4A) whilst the impurities having high hydrophobicity bound tightly and eluted later (FIG. 4B) as in the case with Phenyl-Sepharose.

The procedure was further improved using ammonium sulfate fractionation (AS), HIC/affinity on Phenyl-Sepharose (HIC1), HIC on Butyl-Sepharose (HIC2) and AEX, the active fraction was concentrated and desalted with a molecular sieve with cutoff of 10 kDa. The concentrated and desalted fractions 5 and 6 shown on FIG. 4 were analyzed on SDS-PAGE (FIG. 5). The protein bands were isolated from the gel and digested with trypsin. The digest was separated on RP-HPLC and analyzed by MS to identify the identity of the protein bands.

The MS results indicate that after 4 steps of purification the OTA degrading activity was not confined to one single band, instead the following enzymes were identified (order of intensity): Formamidase (51% sequence coverage), formamidase degradation product (caused by protease) (30%), unannotated A. niger protein sequence (An14g02080, XP_(—)001400834) having similarity to bacterial amidase (25%), glutamyltranspeptidase (17%), and aldose 1-epimerase (14%). From these results the identified enzyme that most likely breaks down OTA was considered to be Aspergillus niger formamidase as indicated by the results in FIGS. 3 and 5, followed by an unannotated A. niger protein sequence having similarity to bacterial amidase and glutamyltranspeptidase, all of the three having activities toward C—N bond, which exists in OTA molecule.

Example 4 Characterization of the Enzymes Identified that May have the Potential to Break OTA Down

Based on the results from FIGS. 3, 4, 5 and 6, further studies were undertaken on the two putative Aspergillus enzymes formamidase and glutamyltranspeptidase, and the hypothetical protein An14g02080 (XP_(—)001400834).

The putative formamidase of Aspergillus origin has not previously been characterized with respect to its biochemical properties including substrate specificity though it had been cloned from A. nidulans and expressed as indicated indirectly by the activity of beta-galactosidase activity (Fraser et al., 2001). Formamidase is known to be involved in glyoxylate and dicarboxylate metabolism and also nitrogen metabolism. It converts formamide in the presence of water to formic acid (formate) and ammonium (formamide+H₂O→formate+NH₃). The presence of formamidase activity was assayed using Megazyme formic acid kit as indicated in the Material and method section above.

When this assay was carried out with fractions obtained in the AEX step, formamidase was observed in fractions of 25 to 30 (FIG. 6A). This was the first indication that fungi and fungal preparations including the Amano™ lipase product prepared from A. niger had an active formamidase breaking down formamide to formic acid and ammonium. Equally important the formamidase activity was purified using the purification steps designed to purify OTA activity, suggesting that fungal formamidase breaks down OTA. When the same fractions tested for formamidase activity were assayed for degradation of OTA, the conversion increased from fraction 26 to 32 (FIG. 6B). As can be seen from FIG. 6A and FIG. 6B the two activities were present in the same fractions. From the data it was further hypothesised that the A. niger formamidase or Aspergillus formamidase that had not been characterized before had OTA degradation activities.

Example 5 Analysis of Formamidase and OTA Degradation Activity in A. niger Transformants Harboring the A. nidulans Formamidase Gene

A. niger formamidase transformants were analyzed for formamidase activity and OTA degrading activity. As can be seen from Table 2 formamidase activity was detected in 12 out of 15 transformants. Surprisingly the formamidase A. niger was apparently not able to break down OTA as can be seen in the transformants that had high formamidase activity but low or no OTA degrading activity. This indicates that fungal formamidase may not have OTA degradation activity but had been co-purified with the OTA activity through the 4 purification steps.

Interestingly by integrating the foreign DNA as in the case of A. nidulans formamidase gene into the A. niger genome the inventors have succeeded by unknown mechanism in creating transformants having higher OTA degrading activities compared to the parent strain used for the transformation A. niger see for example transformants 7#, 2#, 8#, 11#, 15#. Note that 16# was wild type (parent strain). Control was the reaction without the addition of broth. Fermentation broth was used for all these assays. The reaction mixture contained 10 μl broth, 50 μl OTA (1 μg/ml), 100 μl sodium phosphate (67 mM, pH7 or pH9). For control, 10 μl broth from 5-day old culture in a shake flask fermentation was replaced with 10 μl water. At the end of the reaction 160 μl acetonitrile was added to stop the reaction, and filtered and 10 μl injected for HPLC analysis.

Based on the surprising discovery that formamidase of Aspergilli was not able to breakdown OTA the inventors undertook further research to identify if a second candidate for OTA degradation, i.e. the hypothetical A. niger protein (An14g02080, XP_(—)001400834) designated amidase 2 herein had OTA degrading activity.

Example 6 Construction of a Putative Amidase 2 Gene Expression Plasmid

The putative amidase 2 gene from Aspergillus niger encodes the amidase protein of 480 amino acids (SEQ ID NO: 1). To construct the recombinant expression plasmid for A. niger amidase 2 gene, two primers GGAGATCTATCATGGTCCGCCGAATTG and AATCTAGACTAGTGATGGTGATGGTGATGCAGAAAAGGATTACGTG were used in a Pfu Ultra II PCR reaction with genomic DNA template obtained from A. niger UVK143 strain (Ward et al., Appl. Microbial. Biotechnol., 39: 738-743, 1993). The PCR reaction was performed for 30 cycles of 95° C. for 30 seconds, 50° C. for 30 seconds and 72° C. for 1 minute with the two primers. The final extension at 72° C. was done for 5 minutes and the reaction was chilled to 4° C. The nucleotide sequence of the coding region of the resulting PCR amplicon, without the His tag, is listed as SEQ ID NO: 2. The PCR fragment was purified with a Qiagen spin column. It was digested with restriction enzyme BgIII and XbaI and cloned into the pGAPT plasmid vector (see e.g., U.S. Pat. No. 6,426,410) that had been digested with BgIII and XbaI. The resulting plasmid, pGAPT-amidase 2 (shown in FIG. 7) was confirmed by DNA sequencing to have a recombinant gene comprising A. niger amidase 2 inserted between the A. niger glucoamylase promoter and an A. tubingensis glucoamylase terminator. The corresponding encoded protein sequence is shown in SEQ ID No. 4 which is identical to SEQ ID No. 1 except that six histidine residues were added at the c-terminal end of the protein to aid protein purification.

Example 7 Construction of a Recombinant Amidase 2 Production Strain

The AP4 strain of A. niger (Berka et al, Gene 86: 153-162, 1990) was transformed with the pGAPT-amidase 2 plasmid using a PEG-mediated protoplasts fusion transformation protocol. The transformation protocol utilized a modification of the Campbell method (see, Campbell et at., Curr. Genet. 16:53-56 (1989)) with Lysing Enzyme from Trichoderma harzianum (Sigma, L1412). More than one hundred transformants were obtained and thirty transformants were selected on MM plate. The control AP4 parental strain and fifteen transformants were chosen to be grown in CMA plates first and the mycellium agar plugs containing both mycellium and spores from CMA plates were transferred to the shake flasks containing 30 ml of culture medium. The transformants were grown for 6 days at 28° C. The mycellium pellets from control AP4 strain and all fifteen transformants were washed with water at least twice. The mycellium pellets were suspended in CelLytic Y Cell Lysis Reagent (Sigma, C4482). The intracellular proteins were extracted at room temperature for 2 hours. The suspensions were spun down and 15 μl of the supernatants containing extracted intracellular proteins were analyzed using SDS-PAGE gels (FIG. 8). The culture broths were spun down in a microfuge for 10 minutes and 15 μl of the supernatant (extracellular proteins) were analyzed using SDS-PAGE gels (FIG. 8).

TABLE 2 Analysis of formamidase and OTA degradation activity in A. niger transformants harboring the A. nidulans formamidase gene A. niger Transformant No. # harboring the A. nidulans gene 16 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 Wt Control OTA Peak area, at pH 9,  30  7 23 13  27  20 0  7  10 115 7 37 70 108 8.2 36 168 19 hr, 37° C. OTA Peak area, at pH 7,  —* — — — — — — — — 102 — 0 145 138 0 20 128 19 hr at 37° C. OTA Peak area, at pH 9, — — 70 55 — — 93 — — 105 52 86 129 116 — 117 116 45° C. 4 hr OTA Peak area of OTA, — — 17 17 — — 4.3 — — 104 16 14 118 122 — 76 126 at pH 7, 45° C. 4 h Formamidase activity 216 23  0  0 325 160 195 104 129 108 4 431 232 0 171 0 0 at pH 7 at 37° C. *not analyzed

Table 3 shows the medium used (per liter) for growing Aspergillus niger and its transformants in 250 ml siliconized flasks with 30 ml of medium. The A. niger strains were first grown in CMA plates for 3-5 days and 2 cm² plus were transferred to flask and grown at 30° C. for 5 days. For the wild type (control strain or parental strain 620), 0.5 mg/ml of uridine was supplemented.

Corn Streep Solids 50 g NaH₂PO₄•H₂O 1 g MgSO₄•7H₂O 1.0238173 g 50% (w/v) Staley 7350 Corn Syrup 100 mL Sodium Citrate Dihydrate 8 g (Na₃C₆H₅O₇2H₂O) pH to 5.8-6.0 Bring to volume with milliQ H₂O.

Strain can be grown in CMA plate (per liter):

Dextrose (Glucose) 20 g BD Bacto Malt Extract 20 g BD Bacto Peptone  1 g BD Bacto Agar 20 g Autoclave and poor plates

FIG. 8. The SDS-PAGE analysis of intracellular proteins of A. niger harboring the Amidase 2 gene. The protein band of amidase 2 was indicated by an arrow bar. M. molecular markers, AP4, the parent strain of A. niger. The transformants were numbered from 1 to 15.

FIG. 9 shows SDS-PAGE of extracellular proteins of A. niger harboring the Amidase 2 gene. The protein band of amidase 2 is indicated by an arrow bar. The other major bands above the amidase 2 band were α-amylase and glucoamylase secreted by A. niger. The samples loaded to the gels were the fermentation brothes of the different transformants.

In order to confirm the supposed amidase 2 band separated on SDS-PAGE was indeed the product of the amidase gene, the band was cut out, digested with trypsin by in-gel digestion. The digests were separated by RP-HPLC and analyzed by mass spectrometry. This revealed 4 amino acid sequences which are found in the putative amidase 2 sequence (SEQ ID NO: 1) (FIG. 18A). The four sequences identified were:

1. EALQNGY; 2. ALPAGEVLGSY; 3. GAGPL; 4. SVGPQAPL.

Example 8 Characterization of Recombinant Amidase 2 with Respect to its Degradation of OTA and Biochemical, Catalytical Parameters

As shown in FIG. 10, both the intracellular and extracellular fractions (broth) of the transformants of A. niger harboring the putative protein coding gene (amidase 2) plus the parent strain (wt) were assayed for their capability to breakdown OTA. FIG. 10 surprisingly shows that the hypothetical protein encoding A. niger gene from Genbank (An14g02080, Acc No. XP_(—)001400834) encodes an active enzyme having OTA degrading activity and this active enzyme was found both intracellularly and extracellularly. One can see the best transformants are transformant 3, 4, 7, 11, 13 and 14 as no detectable OTA was left (i.e. below the detectable level of 2pbb) after the incubation with the intracellular or the extracellular fractions (the broth). Compared to the parent strain (wt) A. niger, transformant 8, 10 and 15 also had much higher OTA degrading activity.

The recombinant amidase 2 was active at least between pH 3 to 9 in the degrading OTA. FIG. 11 shows that the enzyme activity was the highest at pH 7.0 followed by pH 6 and 8; pH 5 and 9.

The recombinant amidase 2 was apparently heat stable as seen from FIG. 12 as more than 80% activity remained after 5-30 min pre-incubation at 80° C. compared to control where no enzyme was added. The reaction mixture of 5 μl enzyme plus 45 μl 68 mM sodium phosphate (pH7.0) was incubated at 80° C. for 0-30 min before adding another 50 μl the phosphate buffer and 25 μl 1 μg/ml OTA, and incubated at 30° C. for 60 min. The reaction was stopped by adding 130 μl acetonitrile containing 0.2% acetic acid (v/v). The reaction mixture was filtered and injected (10 μl) for HPLC analysis. Ctrl was control where no amidase was added.

Further more the recombinant amidase 2 was quite stable at 70° C. between pH 6 and 9. The stability decreased with the decrease in pH (FIG. 13). Activity was assayed at pH 7.0 and 30° C. The pre-incubation mixture contained 5 μl amidase 2 mixture (155 μg protein/ml), 45 μl 67 mM phosphate buffer (pH3, 4, 5, 6, 7, 8 and 9). The samples were pre-incubated at 70° C. for 30 min. After cooling to 30° C., 2 μl 0.5 mg/ml OTA in ethanol and 80 μl 0.2M Mops-NaOH (pH 7.0) were added and the reaction incubated at 30° C. for 60 min. The reaction was stopped by adding 130 μl acetonitrile containing 0.2% HAC, filtered and injected on RP-HPLC with an injection of 5 μl to monitor the remaining OTA.

Example 9 Purification of the Recombinant Amidase 2 Mat from the Fermentation Broth of A. niger Harboring the Amidase 2 Gene

The broth of transformant 13 which had high OTA activity (FIG. 10) was used to purify the recombinant amidase 2 mat by ammonium sulfate fractionation, affinity chromatography on Phenyl Sepharose, HIC on Butyl-Sepharose and AEX as described below.

1) (NH₄)₂SO₄ fractionation. To 40 ml of the A. niger broth harboring the A. niger amidase 2 gene was added (NH₄)₂SO₄ to 40% saturation by stirring. After 10 min centrifugation at 3500 rpm, the supernatant was collected and (NH₄)₂SO₄ added to 60% saturation by stirring and the solution centrifuged again. The pellet corresponding to the precipitate between 40 to 60% (NH₄)₂SO₄ saturation was dissolved in 40 ml of 50 mM tricine-HCl (pH7.0) containing 1M (NH₄)₂SO₄ and filtered with 0.22 μm filter.

2) Hydrophobic interaction/affinity chromatography. The filtered sample from the above step was loaded a Phenyl Sepharose FF column (2.6×10 cm). Following a wash with tricine-HCl (pH7.0) containing 1M (NH₄)₂SO₄, the active fractions were eluted in 10 ml per tube at a flow rate of 10 ml/min with a linear gradient of 50 mM tricine-HCl (pH 7.0) from 1 M to 0 M of (NH₄)₂SO₄. The purification was achieved on Akta purifier system.

3) Anion exchange chromatography. The active fractions were pooled and desalted on a PD10 column equilibrated in 20 mM Tris/HCl, pH 7.5 (buffer A). The desalted sample was applied to a Source Q15 column equilibrated in buffer A (10 ml/min). The column was washed with buffer A and the bound proteins were eluted with a linear gradient of 0-1 M NaCl in buffer A. During the gradient elution, fractions of 5 ml were collected. The active fractions were analyzed on SDS-PAGE (FIG. 14A), which indicates that the amidase 2 mat was purified basically to homogeneity. The activity was related to a protein band having a molecular mass just below 49 kDa on the SDS-PAGE (FIG. 14). Mass spectrometry by Maldi-tof measurement indicates that this protein band may have a mass of 47214 Da (±2%) (FIG. 14B). N-terminal sequencing of fraction 12 indicate that the N-terminal of the Amidase 2 mat started at STDEAKVTI (SEQ ID NO. 33). The full length of the 480aa amidase 2 with 6 histidine residues is estimated to have a molecular mass of 51,983 Da while the N-terminal 42aa truncated amidase 2 with 6 histidine residues at its C-terminal is supposed to have a mass of 47,338 Da. These indicate that the amidase 2 secreted into the A. niger broth, i.e., amidase 2 mat is the amidase 2 that has been processed by a signal peptidase or other peptidase in the culture broth cleaving between the peptide bond of Ala-Ser in Amidase 2 sig.

Example 10 Substrate Specificity of Amidase 2

Km/Vm Determination:

Using the fractions obtained in the AEX step of amidase 2 mat the kinetic constants of Km and Vm of the enzyme degrading OTA were 0.29 μM/min and 13 μM, respectively by using the Lineweaver-Burk calculation method.

Ochratoxin B Degradation:

The enzyme solution of Amidase 2 mat obtained after AEX was tested on ochratoxin B at the same concentration under optimum conditions for one hour. The degrading rate with ochratoxin B was 8% compared to OTA.

Example 11 The Activity of Pure Amidase 2 mat in Breaking Down OTA

Ochratoxin A (OTA) degrading activity of amidase 2 mat purified from the fermentation broth of A. niger harboring the amidase 2 gene as a function of its concentration is shown in FIG. 15. It can be seen from FIG. 15 that the degradation of OTA was amidase 2 mat concentration dependent. One can also see that after 30 min with an amidase 2 mat concentration of 160 ng/ml the OTA decreased by 83%. From the linear region of FIG. 15 the calculated specific activity for amidase 2 mat was 900 nanomole OTA hydrolyzed per min per mg protein at pH7.0 and 40° C. at an OTA concentration of 1 μg/ml reaction mixture. The reaction for FIG. 15 was performed in 0.1 Mops-NaOH (pH 7.0) and 40° C. for 30 min before it was stopped by adding equal volume of acetonitrile containing 0.2% acetic acid and analyzed on RP-HPLC on a C18 column as described in the Material and Method section.

Example 12 Differences in Sensitivity to Ca²⁺ Between the Intracellular and Extracellular Amidase 2 Found in A. niger

It was observed that the amidase 2 (i.e., amidase 2 mat) purified from the Amano™ lipase product or from the broth of transgenic A. niger harboring the amidase 2 gene were both inhibited by around 40% of its activity in the hydrolysis of OTA by 8 mM CaCl₂ (FIG. 16), while the intracellular fraction of amidase 2 (amidase 2 sig) of transgenic A. niger was not appreciably inhibited by CaCl₂ at 8 mM CaCl₂ (FIG. 17). These results indicate that the loss of the N-terminal of amidase 2 resulted in sensitivity to Ca²⁺ inhibition. The inhibition by Ca²⁺ was overcome by the addition of divalent chelators including EDTA in an equal molar basis. Also it is known that the OTA degrading activity in the Amano™ lipase product is sensitive to EDTA. This example indicates that use of amidase 2 mat in systems containing divalent ions may be aided by the addition of a chelator such as EDTA. Citric acid and other organic acid such as propionic acid are widely used as feed additives and can chelate the divalent ion or trivalent ions, relieving their inhibition of amidase 2 mat. On the other hand extra EDTA also inhibited the recombinant Amidase 2 mat (data not shown). These results suggest that amidase 2 mat can be used for detoxification of OTA but has certain limitations due to its sensitivity to divalent or trivalent metal ions and to chelators like EDTA, while amidase 2 sig was discovered to be much less sensitive to divalent or trivalent metal ions and to chelators like EDTA (see below) indicating the importance of the full length of amidase 2 instead of the secreted or processed or mature amidase 2 (i.e., amidase 2 mat) found in the A. niger broth.

Example 13 Degradation of OTA by the Amidase 2 Extracted from the Intracellular Fraction (Amidase 2 Sig) of A. niger

This example shows the advantage of using the less sensitive form of amidase 2, i.e., amidase 2 sig. As can be seen from FIG. 17, 20 mM CaCl₂ or 20 mM EDTA had no inhibition to the amidase 2 sig compared to amidase 2 mat (FIG. 16). This provides a clear advantage for using amidase 2 sig in additives comprising a chelator in detoxifying OTA. The reaction contained 60 μl mops-NaOH (0.2M, pH7.0), 20 mM CaCl₂, 20 mM EDTA, amidase 2 sig (0.46 μg protein) extracted from A. niger cell harboring the amidase gene, 2 μl 25 μg/ml OTA and water to a final volume of 120 μl. The reaction was performed at 37° C. for 30 min and stopped by adding 150 μl acetonitrile containing 0.2% acetic acid. OTA and its product OT-alpha (=ODα) were analyzed on RP-HPLC on C18 column with an injection of 5 μl. Positive control (PC) contained no additives other than the buffer the amidase and OTA. Blank contained only buffer and OTA.

Example 14 Degradation of OTA in Corn flour and DDGS (Dried Distillers Grains With Solubles) by the Amidase 2 sig Extracted from the Intracellular Fraction (Amidase 2 sig) of A. niger

Corn is the major carrier for OTA and it can be further carried through to and concentrated in DDGS in bioethanol production. Corn and DDGS are widely used as feed ingredients. Mycotoxin level in DDGS is the usual factor that limits the amount of DDGS that can be added to feed. The examples here indicate that ability of amidase 2 sig in degrading OTA in corn flour and DDGS:

For corn flour, 50 mg corn flour was added 210 μl water, 8 μl OTA (25 μg/ml) and 20 μl amidase 2 sig (3.1 μg protein), mixed and incubated at 37° C. for 22 hr. The OTA concentration decreased from 800 ppb at the start to 14 ppb at the end. OTA analysis by HPLC is described in the materials and methods section above.

For DDGS, 1 g was suspended in 10 ml water, pH adjusted to 6.7 with NaOH. To 210 μl of this slurry was added 8 μl OTA (25 μg/ml) and 20 μl amidase 2 sig (3.1 μg protein), mixed and incubated at 37° C. for 22 hr. The OTA concentration decreased from 800 ppb at start to 5 ppb at the end. OTA analysis by HPLC was described in the materials and methods section above.

Example 15 Degradation of OTA in Milk by Amidase 2 mat

It is known that many food products including milk and cheese can be contaminated with OTA. This example tests OTA degradation in milk using amidase 2 mat.

Light milk (1.5% fat, protein 3.4 g, carbohydrate 4.7 g Calcium 120 mg, Phosphorus 95 mg in 100 ml) from Aria Foods Amba (Aarhus, Denmark) 0.15 ml, 3 μl OTA (2.5 μg/ml), 7.5 μl purified amidase 2 mat (from the broth of A. niger harboring the amidase 2 gene) containing 0.22 μg protein were mixed and reacted at 40° C. for 2.5 hr. The OTA decreased from 47 ppb at start to undetectable level (<2 ppb) after 2.5 hr incubation. OTA analysis by HPLC was described in the materials and methods section above.

Example 16 Amidase 2 mat in the Detoxification of OTA in Corn Flour

The reaction mixture in 1.5 ml eppendorf tubes (C1-3) comprised 60 mg corn flower, 200 μl Mops-NaOH (0.2 M pH7.0), 40 μl amidase 2 mixture (which was mixture of an equal volume of the broth of A. niger transformant having amdiase 2 gene 3, 4, 7, 8, 10, 11, 13, 14, and 15), 20 μl OTA (0.5 mg/ml). Total volume of the three components was 260 μl; final OTA was 38 μg/ml or 38 ppm (ppm, parts per million). Control: Tubes of Controls (C-4-6) were the same as C1-3 except that the amidase preparation (the broth) was replaced with water.

The reaction was performed at 30° C. with skaking for 20 hr before the addition of equal volume of acetonitrile 260 μl containing 0.2% acetic acid to stop the reaction and to extract the OTA. After centrifugation and filter, the filtrate was analyzed on HPLC. The HPLC results indicate that the remaining OTA was undetectable (less than 2 ppb), indicating that amidase 2 mat was able to reduce the OTA in corn flour from 38 ppm to less than 2 ppb.

Example 17 Amidase 2 Mat in the Detoxification of OTA in Corn-Soy Based Feed

The tests of amidase 2 mat with soy-corn based feed (whose composition is given in Table 3) contaminated with OTA was exactly as the test with corn flour, that is, tubes S1-3 (experimental) were with the broth while for tubes S 5-6 (controls) water replaced the broth. After 20 hr incubation, the OTA level was reduced from 38 ppm to 6.6 ppm, a reduction of 6 times or 82%.

TABLE 3 The composition of corn/soy based feed used Corn Diet Ingredients Percent Corn 60.01%  Soybean meal 48 31.52%  Soy oil 4.00% Salt 0.40% DL Methionine 0.20% Limestone 1.16% Dicalcium Phos 1.46% VIT/MIN 1.25% Total  100%

Example 18 Solving the crystal structure of Amidase 2

15 crystals of amidase 2 were obtained as shown in Table 4.

Crystals AM7 and AM15 were x-ray diffracted and the structure of these two crystals of amidase 2 (aa 45-480) was solved at 2.5 Å resolution.

Amidase 2 was shown to have a structure similar to a Tim Barrel structure as shown in FIG. 20 and the active site as shown in FIG. 21. A summary of the data for the solved crystal structure for the AM7 crystal set is set out below.

Dataset: am7

Overall InnerShell OuterShell Low resolution limit 80.00 80.00 2.64 High resolution limit 2.50 7.91 2.50 Rmerge 0.182 0.044 0.611 Rmerge in top intensity bin 0.057 — — Total number of observations 441395 14486 65195 Total number unique 46056 1566 6711 Mean((I)/sd(I)) 9.4 20.5 3.3 Completeness 96.5 93.3 97.6 Multiplicity 9.6 9.3 9.7

The structure was solved using Phaser and analyzing an ensemble of 5 most similar structures.

It was found that the active site of amidase 2 (SEQ ID NO:1) includes at least the following residues: His111, His113, His191, Lys246, His287, His289, His307, Asp378 and Val 253. It may accommodate at least two metal ions for catalysis and/structure purposes forming so-called a binuclear metal centre.

The coordinates for one monomer of the enzyme which is in the form of a tetramer are shown in FIG. 22.

TABLE 4 Crystal sets and related parameters Resol./ mol/ Plate/ Beamline/ Set SG cell after scal. Rmerge compl ASU drop Cryst. cond. synchrotron AM1 P2₁2₁2₁ 80 112 173 1.85/1.9 15.5/50.6 95.3/97.4 2-4 AM3/C1 PEG3K, NaAc4.6 ID14-4 ESRF ? AM2 P42₁2 184 184 79 3.19/3.2 16.5/46.4  100/100 2 AM8/B5 PEG3K, K₃cit, Tris 8.5 ID29 ESRF AM3 P42₁2 183.5 183.5 79.5  2.8/3.4 17.4/43.0 99.9/99.6 2 AM8/B6 PEG3K, K₃cit, Bicine 9 ID29 ESRF AM4 ??? ???  2.9/? ?? ?? ?? AM13/C2 AS, Hepes 7 ID29 ESRF AM5 P42₁2 184 184 79  2.9/3.2 17.4/56.5 99.8/99.4 2 AM8/B6 PEG3K, K₃cit, Bicine 9 ID29 ESRF AM6 P42₁2 184 184 79 2.81/3.5 22.3/50.9 99.7/100 2 AM8/A5 PEG3K, K₃cit, Tris 8.5 ID29 ESRF AM7 P42₁2 184 184 80  2.5/2.5 18.2/61.1 96.5/97.6 2 AM8/A5 PEG3K, K₃cit, Tris 8.5 ID29 ESRF AM8 I2 213 80 218  2.7/~3 13.1/66.1 85.8/85.8 8 AM5/A5 PEG3K, K₃cit ID29 ESRF AM9 P42₁2 183 183 94 2.57/3.1 30.3/84.6 86.7/86.7 2 AM12/B5 PEG3K, K₃cit, Tris 8.5 ID14-4 ESRF 4MF AM10 P42₁2 183 183 94  2.7/2.7 16.3/64.7 94.9/97.8 2 AM8/A6 PEG3K, K₃cit, Bicine 9 ID14-4 ESRF 4MF AM11_Zn P2₁2₁2₁ 79 111 172.5 ~2.5 ?? ?? 2-4 AM3/C5 PEG3K, NaAc4.6 ID29 ESRF ? Zn AM12 P42₁2 184 184 79  2.5/2.95 18.3/62.9 91.5/92.8 2 AM15/C6 PEG3K, K₃cit, Bicine 9 ID29 ESRF Zn + 4MF AM13 P42₁2 184 184 79 AM15/C6 PEG3K, K₃cit, Bicine 9 ID29 ESRF Zn + 4MF AM14 P42₁2 113.5 183.5 79  2.5/3.0 19.7/74.6 96.2/97.2 2 AM15/C6 PEG3K, K₃cit, Bicine 9 ID29 ESRF Zn + 4MF AM15 I2 213 80 218 2.45/2.5 10.1/35.8 91.0/70.5 8 AM15/B1 PEG3K, K₃cit, Cit 5 ID29 ESRF

TABLE 5 The structural elements of amidase 2

Color coding: white = small sandwich domain, Dark gray = catalytic barrel domain, light gray = barrel strands and barrel helices; helix α7 may also be considered a barrel helix

Example 19 Expression of Amidases from Fungal Species Glomerella qraminicola M1.001 and Metarhizium anisopliae ARSEF 23 in Trichoderma reesei

To express amidases from Glomerella graminicola M1.001 and Metarhizium anisopliae ARSEF 23, both genes were synthesized as codon optimized sequences for expression in T. reesei and cloned in pDonor221 vector (Invitrogen, Carlsbad, Calif., USA) by Life technologies (Germany). 6×His codons were added at the 3′ coding region of each gene for purification purposes. To enable the expression of the both amidases in Trichoderma reesei, their coding sequences were cloned into the Gateway compatible destination vector pTTT-pyrG13 (described in WO2010141779A1 and PCT/US10/57531) via the Gateway® LR recombination reaction. This vector contains the T. reesei cbhl-derived promoter and terminator regions allowing for a strong inducible expression of a gene of interest, the Aspergillus nidulans amdS and pyrG selective markers conferring growth of transformants on either acetamide as a sole nitrogen source or in the absence of uridine or both, and the T. reesei telomere regions allowing for non-chromosomal plasmid maintenance in a fungal cell. The cbhl promoter and terminator regions are separated by the chloramphenicol resistance gene, Cm^(R), and the lethal E. coli gene, ccdB, flanked by the bacteriophage lambda-based specific recombination sites attR1, attR2. Such configuration allows for direct selection of recombinants containing a gene of interest under the control of the cbhl regulatory elements in the correct orientation via the Gateway® LR recombination reaction.

LR recombination reactions between the pEntry clones of each amidase gene and the destination vector pTTT-pyrG13 were done using the LR Clonase™ II enzyme mix according to the protocol from Invitrogen. Recombination products generated were transformed to E. coli Max Efficiency DH5α, as described by the supplier (Invitrogen), and clones containing the expression constructs pTTT-pyrG13-Amidase G. graminicola and pTTT-pyrG13-Amidase M. anisoplia were selected on 2xYT agar plates (16 g/L Bacto Tryptone (Difco, USA), 10 g/L Bacto Yeast Extract (Difco, USA), 5 g/L NaCl, 16 g/L Bacto Agar (Difco, USA)) with 100 μg/ml ampicillin. After growth of bacterial cultures in 2xYT medium with 100 μg/ml ampicillin, isolated plasmids were subjected to restriction analysis and clones with correct restriction pattern were used for transformation of the Trichoderma reeei 2830 pyrG negative strain deleted for 4 major cellulases cbhl, cbhll, egll, eglll (WO2010141779A1). The final expression plasmids pTTT-pyrG13-Amidase G. graminicola and pTTT-pyrG13-Amidase M. anisopliae are shown in FIG. 23-25.

0.5-2 μg of each expression plasmid was transformed in T. reesei using PEG-Protoplast method with slight modifications, as indicated. For protoplasts preparation, spores were grown for 16-24 hours at 24° C. in Trichoderma Minimal Medium MM (20 g/L glucose, 15 g/L KH₂PO₄, pH 4.5, 5 g/L (NH₄)₂SO₄, 0.6 g/L MgSO₄x7H₂O, 0.6 g/L CaCl₂x2H₂O, 1 ml of 1000×T. reesei Trace elements solution {5 g/L FeSO₄x7H₂O, 1.4 g/L ZnSO₄x7H₂O, 1.6 g/L MnSO₄xH₂O, 3.7 g/L CoCl₂x⁶H₂O}) with shaking at 150 rpm. Germinating spores were harvested by centrifugation and treated with 50 mg/ml of Glucanex G200 (Novozymes AG, Switzerland) solution for lyses of the fungal cell walls. Further preparation of protoplasts was performed by a standard method, as described by Penttiläet al. [Gene 61 (1987) 155-164].

In general, transformation mixtures containing appx 1 μg of DNA and 1−5×10⁷ protoplasts in a total volume of 200 μl were treated with 2 ml of 25% PEG solution, diluted with 2 volumes of 1.2M sorbitol/10 mM Tris, pH7.5/10 mM CaCl₂ solution, mixed with 3% selective top agarose MM containing 1 M sorbitol and 20 mM acetamide (the same Minimal medium as mentioned above but (NH₄)₂SO₄ was substituted with acetamide) and poured onto 2% selective agarose with acetamide. Plates were incubated for 5-7 days at 28° C. before transformants grew and started sporulation.

A spore mixture (10⁶ spores/ml) harvested from a transformation plate was used to inoculate shake flasks with production medium (per 1 L): Glycine production medium (4.7 g/L (NH₄)₂SO₄, 33 g/L 1,4-Piperazinebis(propanesulfonic acid), pH 5.5, 6.0 g/L glycine, 5.0 g/L KH₂PO₄, 1.0 g/L CaCl₂x2H₂O, 1.0 g/L MgSO₄x7H₂O, 2.5 ml/L of 400×T. reesei trace elements, 20 g/L Glucose, 6.5 g/L Sophorose). As a control, T. reesei recipient 2830 strain was grown under the same conditions but in the presence of 10 mM uridine. After 5 days of fermentation at 30° C. and 200 rpm, cultures were harvested and subjected to enzymatic analysis from both extracellular and intracellular extracts.

Example 20 Expression of Aspergillus oryzae Amidase in Aspergillus niger GICC#2445 Strain

To express putative amidase gene from A. oryzae in Aspergillus niger, the amidase encoding sequence was amplified from the Aspergillus oryzae RIB40 genomic DNA (obtained from the Fungal Genetics Stock Collection, FGSC, USA) using the gene specific primers with attB1 and attB2 sites at the 5′ and 3′ termini for subsequent cloning via a Gateway approach. In addition the reverse primer contains 6×His encoding triplets to facilitate purification of the protein expressed.

The following set of primers was used:

Forward 5′ GGGACAAGTTTGTACAAAAAAGCAGGCTCACCATGCACCGGATGCTCAC AGACGACAGAC3′ Reverse 5′ ACCACTTTGTACAAGAAAGCTGGGTCTAGTGGTGGTGGTGGTGGTGTGA TTTAATCAGCATGCCGCCACGC 3′.

All PCR reactions were amplified with a high fidelity Phusion DNA polymerase (Finnzymes OY, Espoo, Finland) under standard conditions recommended by the supplier. After separation of the amplified DNA fragment on a 0.8% agarose gel, it was purified with a Nucleospin® Extract PCR clean-up kit (Macherey-Nagel GmbH & co. KG, Duren, Germany) and 150 ng were recombined with the pDonor221 vector (Invitrogen, Carlsbad, Calif., USA) according to recommendation of the supplier. E. coli DH5α colonies with pEntry clones containing the A. oryzae amidase were selected on 2xYT agar plates with 50 μg/ml kanamycin. Plasmids isolated from bacterial cells were analyzed by their restriction digestion pattern for the insert presence and checked by sequence analysis using a ABI3100 sequence analyzer (Applied Biosystems). The resulting pEntry-Amidase A. oryzae plasmid was used for cloning into a destination vector pRAXdest2, as described in U.S. Pat. No. 7,459,299, via subsequent Gateway LR reaction according to the protocol from Invitrogen (Carlsbad, Calif., USA). Recombination products formed were transformed to E. coli Max Efficiency DH5α, as described by the supplier (Invitrogen), and clones containing the expression construct pRAX-Amidase A. oryzae (FIG. 25) were selected on 2xYT agar plates (16 g/L Bacto Tryptone (Difco, USA), 10 g/L Bacto Yeast Extract (Difco, USA), 5 g/L NaCl, 16 g/L Bacto Agar (Difco, USA)) with 100 g/ml ampicillin. After growth of bacterial cultures in 2xYT medium with 100 μg/ml ampicillin, isolated plasmids were subjected to restriction analysis followed by sequencing using a ABI3100 sequence analyzer (Applied Biosystems). Besides a bacterial sequence, the final expression plasmid pRAX2-Amidase A. oryzae contains the A. oryzae amidase coding region under the control of the A. niger glucoamylase promoter and terminator, the Aspergillus nidulans pyrG gene as a selective marker and the A. nidulans AMA1 sequence for autonomous replication in fungal cells.

0.5-2 μg of this plasmid was transformed in A. niger var awamori GICC#2445 strain developed by Genencor using a common transformation procedure known in the art or described in U.S. Pat. No. 7,459,299. This strain is deleted for the endogenous glucoamylase glaA gene and carries a mutation in the pyrG gene allowing for selection of transformants for uridine prototrophy. A. niger transformants were grown on MM medium (the same minimal medium as was used for T. reesei transformation but 10 mM NH₄Cl was used instead of acetamide as a Nitrogen source) for 4-5 days at 37° C. and a total population of spores (10⁶ spores/ml) from different transformation plates was used to inoculate shake flasks with production medium (per 1 L): 12 g Trypton; 8 g Soyton; 15 g (NH₄)₂SO₄; 12.1 g NaH₂PO₄xH₂O; 2.19 g Na₂HPO₄x2H₂O; 1 g MgSO₄x7H₂O; 1 ml Tween 80; 150 g Maltose; pH 5.8. After 5 days of fermentation at 30° C. and 200 rpm, cultures were harvested and subjected to enzymatic analysis from both extracellular and intracellular extracts.

Example 21 Amidases from Three Additional Fungi for Ochratoxin Degradation

The putative amidase sequences from Glomerella graminicola (GMGM), Metarhizium anisopliae and Aspergillus oryzae all showed activity in degrading ochratoxin A.

Table 6 shows the activity of three fungal amidases from Glomerella graminicola, Metarhizium anisopliae and Aspergillus oryzae expressed intracellularly in Trichoderma reesei and Aspergillus niger. The reaction mixture using OTA as substrate consisted of 75 μl phosphate (67 mM, pH7), 5 μl OTA (2.4 μg/ml) and 20 μl intracellular cell-free fraction. The reaction was performed at 23° C. for 17 h and stopped by adding 0.15 ml acetonitrile and analyzed on HPLC with an injection volume of 5 μl.

TABLE 6 Trichoderma Trichoderma reesei parent reesei parent Aspergillus strain strain niger strain transformed transformed transformed Trichoderma with putative with putative with putative reesei parent amidase gene amidase gene amidase strain used for from from gene from the Glomerella Metarhizium Aspergillus Strains/and activities transformation gramicola anisopliae oryzae Activity on ochratoxin A 12.26 0.68 0.96 8.57 expressed as ochratoxin A peak area

The data in the table indicate that the amidase from C. gramicola has the highest activity against ochratoxin, whereas the amidase from A. oryzae has lower activity against this substrate.

Example 22 Sequence Comparison of Putative Amidase Sequences

Sequence comparisons were done over the full length of the polypeptide sequences using the AlignX Vector NTI program (InvitrogenCorporation) using the default parameters (Gap opening penalty−10, Gap extension penalty 0.1). As can be seen in Table 7 the putative amidases may have low overall homology. However, as can be seen from FIG. 26 all of the polypeptides of SEQ ID NOs: 5-11, 13, 14 and 15 have the same motifs as amidase 2 (SEQ ID NO:1 and 3).

The sequences are aligned with Multalin version 5.4.1 (F. CORPET, 1988, Nucl. Acids Res., 16: 10881-10890). Parameters used: blosum62, Gap weight: 12, Gap length weight: 2, Consensus levels: high=90% low=50%, Consensus symbols: ! is anyone of IV, $ is anyone of LM, % is anyone of FY, # is anyone of NDQEBZ. Descriptions of the sequences and their web accession number (in parenthsis):

Furthermore, as shown in Example 21 (Table 6) SEQ ID NO:13-15 have ochratoxin degrading activity.

TABLE 7 Sequence ID NO % identity to SEQ ID NO: 1 (full length) SEQ ID NO: 5 41.8% SEQ ID NO: 6 42.4% SEQ ID NO: 7 42.4% SEQ ID NO: 8 40.7% SEQ ID NO: 9 36.5% SEQ ID NO: 10 28.2% SEQ ID NO: 11 36.5% SEQ ID NO: 13 56.1% SEQ ID NO: 14 53.4% SEQ ID NO: 15 34.6%

The common sequence motifs of the 11 sequences have the Consensus:

1) l/m-P-G-l/m-w/l-D-c/v/s/a-H-x-H-f/y/l-xG, where the two His residues are in the active site;

2) G-y/f-T;

3) G-t/a/s/v/r-i/f/a-x-G-P;

4) G-H-g/s-D where the His residue is in the active site;

5) D-G-v/e-x-e/d/g-C-x-x-a/g/t-v/a-R-x-q/m/a-l/i/v-R-r/h/c-g/n-A-k/r/t/e/d-x-l-K, where the Lys residue is in the active site

6) G-G-V-l/m/v/g-S-x-x-D-x-P, where the Val residue is in the active site;

7) V-a/s/h-A-H-c/v/a-h/q-G-k/r-x-G, where the two His residues are in the active site;

8) H-g/v/a-s/t/l-y/f/e-l/a/l-D, where the His residue is in the active site;

9) G-V-x-l-a/v-I/a-G-T-D, where Asp residue is in the active site.

SEQ ID NO:1, is amidase 2 from A. niger, SEQ5, hypothetical protein from A. niger (XP_(—)001400981), SEQ ID NO:6, hypothetical protein from A. flavus (XP_(—)002385313), SEQ ID NO:7, hypothetical protein from Talaromyces stipitatus (XP_(—)002477972), SEQ ID NO:8, hypothetical protein from Neurospora crassa (XP_(—)962238), SEQ ID NO:9, putative amidohydrolase from Streptomyces roseosporus (ZP_(—)04712156), SEQ ID NO:10, putative amidohydrolase from Thermotoga lettingae (YP_(—)001470371), SEQ ID NO:11, putative amidohydrolase from Salinispora arenicola (YP_(—)001536995), SEQ ID NO:13, putative amidohydrolase from Glomerella graminicola (EFQ25792), SEQ ID NO:14, putative amidohydrolase from Metarhizium anisopliae (EFZ00058), SEQ ID NO:15, hypothetical protein from Aspergillus oryzae (XP_(—)001826758).

Example 23 Sequence Comparison of SEQ ID NO:1 to Carboxypeptidase Having Ochratoxin Degrading Activity

Two carboxypeptidases are known which have low ochratoxin degradding activity, namely bovine carboxypeptidase A and yeast carboxypeptidase Y (Luis Abrunhosa, Robert R. M. Paterson and Armando Venancio, Biodegradation of Ochratoxin A for Food and Feed Decontamination, Toxins 2010, 2, 1078-1099).

The ochratoxin degrading activity of these enzymes has been confirmed. However, as can be seen from FIG. 27, these enzymes show no homology to SEQ ID NO:1 and do not contain any of the 9 motifs associated with ochratoxin degrading ability. Furthermore, these enzymes do not have the amidase typeTim Barrel structure.

SEQUENCE LISTING SEQ ID NO: 1 Amidase 2 sig from A. niger   1 MVRRIASATP MVQSPMSPLG TTYCVRPNPV SLNLQRRPLV IASTDEAKVT  51 IIYAGLLIPG DGEPLRNAAL VISDKIIAFV GSEADIPKKY LRSTQSTHRV 101 PVLMPGLWDC HMHFGGDDDY YNDYTSGLAT HPASSGARLA RGCWEALQNG 151 YTSYRDLAGY GCEVAKAIND GTIVGPNVYS SGAALSQTAG HGDIFALPAG 201 EVLGSYGVMN PRPGYWGAGP LCIADGVEEV RRAVRLQIRR GAKVIKVMAS 251 GGVMSRDDNP NFAQFSPEEL KVIVEEAARQ NRIVSAHVHG KAGIMAAIKA 301 GCKSLEHVSY ADEEVWELMK EKGILYVATR SVIEIFLASN GEGLVKESWA 351 KLQALADSHL KAYQGAIKAG VTIALGTDTA PGGPTALELQ FAVERGGMTP 401 LEAIKAATAN APL SVGPQAP L TGQLREGYE ADVIALEENP LEDIKVFQEP 451 KAVTHVWKGG KLFKGPGIGP WGEDARNPFL SEQ ID NO. 2 Sequence type: DNA; DNA name: DNA containing amidase 2 gene, Length: 1443, Organism: Aspergillus niger ATGGTCCGCC GAATTGCTTC AGCTACACCT ATGGTGCAAT CGCCCATGTC GCCATTGGGC ACAACATACT GCGTCCGTCC TAATCCTGTT TCACTGAATC TTCAAAGAAG ACCTCTCGTG ATCGCATCAA CAGACGAGGC CAAGGTCACT ATAATATATG CCGGACTATT AATCCCTGGC GACGGAGAAC CTCTGCGCAA TGCTGCCCTA GTCATCAGCG ATAAGATCAT CGCGTTCGTT GGATCCGAAG CCGACATCCC TAAGAAATAC CTCCGGTCCA CGCAGTCTAC TCATCGTGTC CCCGTGCTCA TGCCTGGTTT GTGGGATTGC CACATGCATT TTGGCGGGGA TGACGATTAT TACAACGATT ATACATCTGG TCTGGCCACT CATCCAGCAT CATCAGGTGC TCGACTAGCC CGTGGTTGCT GGGAAGCATT GCAGAATGGG TATACATCCT ACCGCGACCT AGCCGGATAC GGGTGCGAGG TCGCAAAGGC GATCAATGAT GGCACTATCG TTGGTCCAAA CGTGTACTCG TCTGGCGCTG CACTCAGTCA GACAGCTGGA CACGGCGATA TCTTCGCTCT TCCAGCAGGC GAAGTACTGG GGAGTTATGG AGTAATGAAC CCACGCCCTG GGTACTGGGG GGCAGGGCCG CTATGTATCG CCGATGGCGT AGAGGAGGTC CGACGAGCAG TGAGGTTGCA GATCCGTCGC GGTGCAAAGG TTATCAAAGT GATGGCCTCT GGGGGTGTCA TGTCGCGAGA CGATAATCCC AACTTTGCAC AGTTCTCTCC AGAAGAACTG AAGGTGATAG TGGAAGAGGC GGCTCGACAG AACCGGATCG TTTCTGCACA TGTGCATGGC AAGGCGGGGA TTATGGCTGC TATCAAAGCA GGCTGCAAGA GTCTGGAGCA TGTGTCTTAT GCTGACGAGG AGGTCTGGGA GCTCATGAAA GAGAAGGGAA TTTTGTATGT GGCCACACGC TCGGTTATTG AAATCTTTCT GGCTAGTAAT GGAGAGGGGT TGGTGAAAGA GTCGTGGGCC AAGTTGCAGG CCCTTGCCGA TTCGCATTTG AAAGCTTATC AGGGAGCTAT TAAGGCGGGT GTTACCATTG CGTTGGGAAC GGATACCGCC CCCGGTGGTC CTACCGCACT TGAGTTGCAG TTTGCCGTCG AGAGAGGAGG TATGACGCCG TTGGAGGCCA TCAAAGCCGC AACTGCGAAC GCTCCCCTGT CAGTTGGTCC ACAAGCACCG TTGACGGGTC AGCTTCGCGA GGGGTATGAG GCAGATGTGA TTGCGTTGGA GGAGAATCCA TTGGAGGACA TCAAAGTCTT TCAGGAGCCG AAGGCAGTTA CCCACGTCTG GAAGGGAGGG AAACTGTTCA AAGGTCCAGG TATTGGTCCG TGGGGAGAAG ATGCACGTAA TCCTTTTCTG TAG SEQ ID NO. 3 Sequence type: Protein; Protein name: amidase 2 mat, Length: 438, Organism: Aspergillus niger SerThrAspGluAlaLysValThrIleIleTyrAlaGlyLeuLeuIleProGlyAspGly GluProLeuArgAsnAlaAlaLeuValIleSerAspLysIleIleAlaPheValGlySer GluAlaAspIleProLysLysTyrLeuArgSerThrGlnSerThrHisArgValProVal LeuMetProGlyLeuTrpAspCysHisMetHisPheGlyGlyAspAspAspTyrTyrAsn AspTyrThrSerGlyLeuAlaThrHisProAlaSerSerGlyAlaArgLeuAlaArgGly CysTrpGluAlaLeuGlnAsnGlyTyrThrSerTyrArgAspLeuAlaGlyTyrGlyCys GluValAlaLysAlaIleAsnAspGlyThrIleValGlyProAsnValTyrSerSerGly AlaAlaLeuSerGlnThrAlaGlyHisGlyAspIlePheAlaLeuProAlaGlyGluVal LeuGlySerTyrGlyValMetAsnProArgProGlyTyrTrpGlyAlaGlyProLeuCys IleAlaAspGlyValGluGluValArgArgAlaValArgLeuGlnIleArgArgGlyAla LysValIleLysValMetAlaSerGlyGlyValMetSerArgAspAspAsnProAsnPhe AlaGlnPheSerProGluGluLeuLysValIleValGluGluAlaAlaArgGlnAsnArg IleValSerAlaHisValHisGlyLysAlaGlyIleMetAlaAlaIleLysAlaGlyCys LysSerLeuGluHisValSerTyrAlaAspGluGluValTrpGluLeuMetLysGluLys GlyIleLeuTyrValAlaThrArgSerValIleGluIlePheLeuAlaSerAsnGlyGlu GlyLeuValLysGluSerTrpAlaLysLeuGlnAlaLeuAlaAspSerHisLeuLysAla TyrGlnGlyAlaIleLysAlaGlyValThrIleAlaLeuGlyThrAspThrAlaProGly GlyProThrAlaLeuGluLeuGlnPheAlaValGluArgGlyGlyMetThrProLeuGlu AlaIleLysAlaAlaThrAlaAsnAlaProLeuSerValGlyProGlnAlaProLeuThr GlyGlnLeuArgGluGlyTyrGluAlaAspValIleAlaLeuGluGluAsnProLeuGlu AspIleLysValPheGlnGluProLysAlaValThrHisValTrpLysGlyGlyLysLeu PheLysGlyProGlyIleGlyProTrpGlyGluAspAlaArgAsnProPheLeu SEQ ID NO. 4 Amidase 2 A. niger + c terminal 6H MVRRIASATPMVQSPMSPLGTTYCVRPNPVSLNLQRRPLVIASTDEAKVTIIYAGLLIPGDGE PLRNAALVISDKIIAFVGSEADIPKKYLRSTQSTHRVPVLMPGLWDCHMHFGGDDDYYNDYTS GLATHPASSGARLARGCWEALQNGYTSYRDLAGYGCEVAKAINDGTIVGPNVYSSGAALSQTA GHGDIFALPAGEVLGSYGVMNPRPGYWGAGPLCIADGVEEVRRAVRLQIRRGAKVIKVMASGG VMSRDDNPNFAQFSPEELKVIVEEAARONRIVSAHVHGKAGIMAAIKAGCKSLEHVSYADEEV WELMKEKGILYVATRSVIEIFLASNGEGLVKESWAKLQALADSHLKAYQGAIKAGVTIALGTD TAPGGPTALELQFAVERGGMTPLEAIKAATANAPLSVGPQAPLTGQLREGYEADVIALEENPL EDIKVFQEPKAVTHVWKGGKLFKGPGIGPWGEDARNPFLHHHHHH SEQ ID NO. 5 Sequence type: Protein; Protein name: putative amidase 2 homologue, Length: 420, Organism: Aspergillus niger MetHisArgProLeuThrAspSerThrPheTyrArgIleAsnAlaAspIleLeuIlePro GlyArgGlyAlaProIleAlaArgGlyAlaValValTrpLysSerLysThrIleLeuTyr SerGlyProGlnHisGluValProValGluTyrGlnAspAlaValThrThrHisValPro ValAlaMetProGlyMetTrpAspCysHisIleHisPheLeuGlyAlaThrAlaAlaThr MetAspSerIleValAsnThrProGlnAlaLeuAlaGlyAlaArgSerValProTyrLeu TyrAlaThrIleIleAlaGlyPheThrSerValArgGluValGlyGlyTyrGlyCysGlu LeuAlaLysValIleAspGluGlyArgIleProGlyProThrIleTyrGlyAlaHisSer AlaIleSerMetThrAlaGlyHisGlyAspValHisGlyValAsnProGluGlyLeuArg AspLeuCysThrHisGlyLeuProLeuThrIleAlaAspGlyValProGluCysLeuGln AlaValArgLysGlnLeuArgHisGlyAlaArgIleIleLysValCysAlaSerGlyGly ValValSerAlaIleAspAspProGlnHisGlnGluPheSerPheGluGluLeuLysAla IleValAspGluAlaAlaArgAlaArgArgValValAlaAlaHisCysHisGlyLysAla GlyIleMetAsnAlaLeuArgAlaGlyCysArgThrIleGluHisGlySerTyrLeuAsp GluGluAlaIleAspLeuMetLeuGluLysGlyAlaMetLeuValAlaThrArgSerVal IleGluSerGlyLeuAlaMetArgAspLeuPheThrProGlySerTyrGlnLysLeuLeu GluValAlaAspThrHisLysArgAlaTyrGluLeuAlaValArgArgGlyValProIle AlaLeuGlyThrAspGlnPheIleSerSerAspAsnProAlaLeuGlyTyrGlyArgAsn GlyLysGluLeuValTyrAlaValAlaAlaGlyMetThrProLeuAlaAlaIleGluAla AlaThrAlaAsnGlyProLeuThrLeuGlyAspGlnAlaProLysSerGlyGlnLeuArg GluGlyPheAspAlaAspIleIleAlaLeuThrAlaAsnProLeuGluAsnIleIleVal ValSerAspProLysAsnValThrHisValTrpArgTyrGlyLysLeuValLysSerAsn SEQ ID NO. 6 Sequence type: Protein; Protein name: putative amidase 2 homologue, Length: 419, Organism:  Aspergillus flavus MetHisArgMetLeuThrAspAspArgLeuTyrArgValAspAlaAspLeuLeuIlePro GlyLysGlyAspProIleProHisGlyAlaValValTrpGlnCysLysThrIleArgTyr AlaGlyProArgSerGluValProAlaGluPheGlnGlyAlaThrThrThrHisValPro ValValMetProGlyMetTrpAspCysHisIleHisPheLeuGlyAlaThrAlaAlaThr MetAsnAlaIleValAspThrProGlnAlaLeuAlaGlyAlaArgSerValProAspLeu HisAlaThrValMetAlaGlyPheThrSerValArgGluValGlyGlyTyrGlyCysAsp LeuAlaLysAlaValGlyGluGlyArgIleProGlyProAsnIleTyrSerSerHisSer AlaIleSerMetThrAlaGlyHisGlyAspValHisGlyValHisArgAspSerLeuLeu AspLeuCysAlaHisGlyLeuProLeuThrIleAlaAspGlyValProGluCysLeuLeu AlaValArgLysGlnLeuArgArgGlyAlaThrValIleLysValCysAlaSerGlyGly ValValSerAlaIleAspAspProGlnHisGlnGluPheSerPheGluGluLeuLysAla IleValAspGluAlaAlaArgAlaArgArgValValAlaAlaHisCysHisGlyLysAla GlyIleMetAsnAlaLeuArgAlaGlyCysArgThrIleGluHisGlySerPheLeuAsp GluGluAlaValGluLeuMetLysGluLysGlyAlaIleLeuValAlaThrArgSerVal IleGluSerGlyLeuAlaMetLysAspLeuPheThrProSerSerTyrGlnLysLeuLeu GluValAlaAspAlaHisArgLysAlaTyrGlnLeuAlaIleSerArgGlyValThrIle AlaLeuGlyThrAspGlnPheIleSerSerAspAsnProMetIleGlyTyrGlyArgAsn GlyHisGluValArgTyrAlaValAspAlaGlyLeuThrProLeuAlaAlaIleGluAla AlaThrAlaAsnGlyProLeuThrLeuGlyTyrGlnAlaProGlnSerGlyGlnLeuLys GluGlyTyrAspAlaAspIleIleAlaValArgGluAsnProLeuGluAsnValAlaVal LeuSerAsnSerLysAsnValThrHisValTrpArgGlyGlyMetLeuIleLysSer SEQ ID NO. 7 Sequence type: Protein; Protein name: putative amidase 2 homologue, Length: 405, Organism: Talaromyces stipitatus MetArgValArgLeuLysAlaSerIleLeuIleProGlyArgGlyGluProIleGluAsn GlyAlaLeuIleIleAspGlyProLysIleAlaTrpValGlyGlnGlnSerAlaIlePro ThrLysTyrGlnAspValAspPheGluTyrLeuProValLeuMetProGlyLeuTrpAsp CysHisThrHisPheMetGlyLeuSerAspGluSerAspThrMetGlnAlaValPheGly SerAlaAlaLeuAlaGlyAlaIleAlaAlaLysGluLeuGluThrAlaLeuMetAlaGly PheThrThrIleArgGluValGlyGlyValAlaGlyGluIleTyrProAlaIleLysAsn GlyThrIleValGlyProAsnValTyrSerSerIleGlyValLeuGlyIleThrGlyGly HisSerAspValHisAsnValProIleGluAlaValIleAlaLysArgAsnGluGlyThr PheValValCysAspGlyValSerAspCysIleLysThrValArgMetMetValArgArg GlyAlaThrLeuIleLysIleCysAlaThrGlyGlyValGlySerLeuLeuAspAspPro GluAspAlaGlnPheSerProGluGluIleLysAlaIleValAspGluAlaAlaArgSer LysArgIleValAlaAlaHisCysHisGlyLysGluGlyIleMetAsnAlaLeuHisAla GlyValHisThrIleGluHisGlySerTyrLeuAspGluGluValAlaAlaLeuMetLys GluLysLysAlaLeuPheValSerThrArgLeuIleIleGluGluGlyLeuLysAsnPro LysLeuTrpProProSerSerTyrArgLysLeuThrLysIleSerGluAlaHisLysLys AlaTyrAlaLeuAlaValLysSerGlyValLysIleValLeuGlyThrAspTrpThrAla GlyGluAsnGlyLysGluLeuAlaTyrAlaValGluAlaGlyMetSerProLeuGluAla IleGluAlaSerThrAlaArgCysProGluThrLeuGlySerHisPheAlaProLeuSer GlyGlnLeuLysGluGlyTyrGlyAlaAspValIleAlaValAlaSerAsnProLeuAsp AspIleLysValLeuGlyGluProLysAsnIleThrHisValTrpLysGlyGlyLysLeu TyrLysGlyThrLeu SEQ ID NO. 8 Sequence type: Protein; Protein name: putative amidase 2 homologue, Length: 440, Organism: Neurospora crassa MetGlnIleLysValThrLeuProAsnAspThrIleAsnArgAspSerValAspAspArg AlaSerTyrHisGlyIleLeuAlaAspValLeuIleProGlyArgGlyGluProLeuLys AsnGlyAlaLeuValValLysAspSerValIleGluTrpValGlyProSerAspGluIle ProSerGluTyrSerSerIleArgValSerArgValProValLeuMetProGlyMetTrp AspValHisThrHisTyrGluGlyValGlyValAlaGlnGlyIleArgGluSerMetLys ProPheLeuProGlyThrAlaThrLeuIleGlyAlaValIleValAspAspMetArgArg ThrLeuMetAlaGlyPheThrSerIleArgGluLeuGlyGlyTyrAlaGlyAspValAla ProAlaIleAspMetGlyAlaIleValGlyProHisValTyrAlaAlaMetSerLeuLeu SerIleThrGlyGlyHisGlyAspLeuHisAspValProLeuArgThrValLeuAspAla CysAlaAsnGlySerSerSerCysPheLeuCysAspGlyValAspGlyCysIleAsnAla ValArgGlnGlnIleArgArgGlyAlaLysValIleLysValCysSerThrGlyGlyVal LeuSerLeuAsnAspGlnProGluAspThrGlnPheSerAlaGluGluLeuArgAlaIle ValGlnGluAlaLysArgSerSerArgValValAlaAlaHisAlaHisGlyLysProGly IleMetAlaAlaLeuAspAlaGlyValLysSerIleGluHisGlySerPheLeuAspGlu GluValAlaAlaLysMetLysGluLysAspAlaIleLeuValProThrArgHisValVal GluGlyMetAlaAlaAsnAsnAspAspLeuAspProArgGlnArgAlaLysLeuGluArg ThrMetGlnLeuSerArgAspSerIleLysLeuAlaHisArgMetGlyValLysIleAla LeuGlyThrAspThrPheArgSerAspLysAsnHisAlaValAlaHisGlyLysAsnAla MetGluLeuArgTyrAlaIleGluAlaGlyMetThrProLeuGlnAlaIleGluMetAla ThrAlaThrProProGluThrLeuGlyProGlnAlaArgLysSerGlyGlnLeuLysAla GlyTyrAspAlaAspLeuIleAlaIleSerSerAsnProLeuGluAspIleGluIleLeu IleAspProAspAsnIleThrHisValTrpLysGlyGlyValLeuPheLysCysProGln SEQ ID NO. 9 Sequence type: Protein; Protein name: putative amidase 2 homologue, Length: 413, Organism: Streptomyces roseosporus MetGluHisArgIleAspAlaAspLeuLeuIleProGlyAlaGlyGluProThrValAsn GlySerValValHisAlaAspGlyArgIleArgPheAlaGlyProThrAlaGluLeuPro ArgGluHisArgAlaLeuGluProThrArgValAlaThrLeuLeuProGlyLeuTrpAsp CysHisValHisPheAlaGlyIleArgGlyArgValSerThrGluGluLeuMetLeuThr ProGluThrLeuAlaValValArgSerValLysAspAlaGluThrAlaLeuArgAlaGly PheThrSerValArgAspMetGlyGlyHisGlyCysValLeuAlaGluAlaValArgGlu GlyThrPheThrGlyProAsnIleTyrSerAlaAsnGlnValIleGlyGlnThrGlyGly HisSerAspAlaHisArgLeuProTyrArgTrpValThrAspProCysArgSerGlyGly ThrLeuArgIleAlaAspGlyValAspGluCysValArgAlaValArgLeuGlnLeuArg AlaGlyAlaGluLeuIleLysIleCysThrSerGlyGlyValLeuSerGluValAspAsn ProValHisGlnGlnTyrArgSerGluGluLeuAsnAlaIleValThrGluAlaAlaArg AlaAspArgValValAlaAlaHisCysHisGlyArgAlaGlyIleLeuAlaAlaIleAsp AlaGlyCysHisThrValGluHisGlyThrGluIleAspGluArgThrAlaAspLeuMet AlaGluArgGlyMetThrLeuValProThrArgThrIleTyrGluAlaPheArgGlnAsp ValAlaAlaLeuProProAlaTrpArgAspArgPheAlaLeuMetAlaGluArgHisLeu ThrAlaIleGlyIleAlaHisArgAlaGlyValThrIleAlaLeuGlyThrAspLeuGly ThrSerAspArgGlyGlyProLeuSerTrpGlyGlyHisGlySerGluPheAlaHisLeu ValSerAlaGlyLeuSerProLeuGluAlaIleLysAlaAlaThrAlaHisGlyProGly ThrLeuGlyProArgAlaProArgSerGlyArgLeuGluAlaGlyTyrAspAlaAspLeu LeuAlaValAspGlyAsnProLeuAlaAspIleThrValLeuAlaAspProAspArgIle ThrArgValTrpLysSerGlyGluProValProSerGly SEQ ID NO. 10 Sequence type: Protein; Protein name: putative amidase 2 homologue, Length: 407, Organism: Thermotoga lettingae GluSerAlaValPheIleGluGlyAlaArgIleLeuAlaValGluLysIleLysArgSer GlnIleProSerGlyPheThrGlnIleAspLeuGlnGlyArgTyrLeuMetProGlyLeu IleAspAlaHisLeuHisLeuAlaGlyMetArgSerGlyAspMetValLysGluHisLeu LeuThrProTyrGluThrLeuValAlaArgThrValThrAspLeuLysSerLeuIleGlu AlaGlyPheThrThrValValAspAlaGlyGlySerIleAlaIleAsnLeuLysLysAla IleGlnGluGlyThrIleAlaGlyProArgIleValAlaAlaGlyHisSerLeuSerGln ThrPheGlyHisGlyAspGluHisPheLeuProIleAspTyrValAspProArgThrSer LysPheLysGlyGlyPheGlySerLeuIleCysAspGlyValAlaGluCysIleLysAla AlaArgTyrAlaLeuArgCysGlyAlaAspPheIleLysIleMetAlaThrGlyGlyVal LeuSerGluArgAspArgProGluTyrThrGlnPheThrValGluGluIleLysAlaIle ValGluGluAlaAsnHisAlaArgLysPheValHisAlaHisAlaGlnGlyLysAspGly IleMetAsnAlaLeuLeuGlyGlyValLysValIleAlaHisAlaIleTyrIleAspAsp GluSerCysLysLeuAlaLysGluLysAsnAlaIleIleValProThrLeuSerIleVal GluHisLeuIleIleHisGlyLysGlnIleGlyAlaProGluTrpGlyLeuArgLysSer GluGluValTyrLysIleHisValGluAsnIleLysLysAlaTyrGluHisGlyValLys IleAlaAlaGlyThrAspPheIleGlyGlyThrLysAlaPheLysHisGlyGluAsnAla LeuGluIleLeuLeuLeuValAspLysIleGlyMetLysProGluGlnAlaLeuLeuSer AlaThrLysValAlaAlaGluAlaAlaGlyLeuSerGlnLeuValGlySerIleAspLys GlyLysLeuAlaAspLeuLeuIleValGluAspAsnProLeuSerAsnValLysIleLeu MetAspHisSerLysIleSerAlaValPheLysGluGlyIleLeuPheLysAspLysIle GlyLeuGluLysTyrPheAsn SEQ ID NO. 11 Sequence type: Protein; Protein name: putative amidase 2 homologue, Length: 408, Organism: Salinispora arenicola MetIleGluCysIleGluAlaAspGlnLeuIleProGlyArgGlyGluProValAlaAsn AlaValValValLeuGluAspAlaThrIleArgTyrAlaGlyProAlaGluHisAlaPro LysValAlaGluAlaArgArgSerArgAlaHisThrValLeuProGlyLeuTrpAspSer HisValHisPheMetGlyLeuArgSerAlaAspValGlyIleLeuProGlnGluProVal AlaLeuArgAlaAlaArgThrValAlaAspLeuArgAlaAlaLeuAspAlaGlyPheThr SerValArgGluValGlyGlyLeuGlyLeuAspLeuAlaArgAlaValGluGluGlyThr AlaValGlyProSerValTyrAlaAlaGlyCysAlaLeuSerThrThrGlyGlyHisGly AspLeuHisSerTyrProLeuAlaTrpMetGluGluPheAlaArgHisGlyGlyGluLeu ArgLeuAlaAspGlyGluAlaGluCysValArgAlaValArgGluGlnLeuArgArgAsn AlaLysValIleLysValTyrAlaSerGlyGlyValLeuSerGluValAspHisProIle HisArgGlnPheThrAspArgGluLeuArgAlaIleValGluValAlaGlyLeuAlaAsp ArgValValAlaAlaHisCysHisGlyLysProGlyMetMetAlaAlaIleGluAlaGly ValArgThrIleGluHisGlyThrTyrLeuAspGluGluValAlaAlaAlaMetArgGlu ThrGlyAlaIleLeuValThrThrArgThrIleMetGlnGluLeuIleAspSerArgAla LeuProProTyrAlaLeuArgLysLeuGluSerIleValAspArgHisAlaGluAlaIle ValIleAlaArgGluSerGlyValArgIleAlaAlaGlyThrAspValAlaLeuThrGly AlaGluLeuProAspSerTrpGlyArgAsnGlyArgGluLeuProLeuLeuAlaGluIle GlyPheSerProLeuGluValIleGluAlaAlaThrAlaAlaAlaProAlaThrLeuGly ProGlnAlaProArgSerGlyGlnLeuValGluGlyTyrAspAlaAspValIleThrLeu AspAlaAspProLeuAlaAspIleGlyValLeuAlaLysProAlaHisIleThrGlyVal TrpLysAlaGlyCysArgValAla SEQ ID NO. 12. Sequence type: Protein; Protein name: pepAd2 (protease), Length: 480, Organism: Aspergillus niger MetHisLeuProGlnArgLeuValThrAlaAlaCysLeuCysAlaSerAlaThrAlaPhe IleProTyrThrIleLysLeuAspThrSerAspAspIleSerAlaArgAspSerLeuAla ArgArgPheLeuProValProAsnProSerAspAlaLeuAlaAspAspSerThrSerSer AlaSerAspGluSerLeuSerLeuAsnIleLysArgIleProValArgArgAspAsnAsp PheLysIleValValAlaGluThrProSerTrpSerAsnThrAlaAlaLeuAspGlnAsp GlySerAspIleSerTyrIleSerValValAsnIleGlySerAspGluLysSerMetTyr MetLeuLeuAspThrGlyGlySerAspThrTrpValPheGlySerAsnCysThrSerThr ProCysThrMetHisAsnThrPheGlySerAspAspSerSerThrLeuGluMetThrSer GluGluTrpSerValGlyTyrGlyThrGlySerValSerGlyLeuLeuGlyLysAspLys LeuThrIleAlaAsnValThrValArgMetThrPheGlyLeuAlaSerAsnAlaSerAsp AsnPheGluSerTyrProMetAspGlyIleLeuGlyLeuGlyArgThrAsnAspSerSer TyrAspAsnProThrPheMetAspAlaValAlaGluSerAsnValPheLysSerAsnIle ValGlyPheAlaLeuSerArgSerProAlaLysAspGlyThrValSerPheGlyThrThr AspLysAspLysTyrThrGlyAspIleThrTyrThrAspThrValGlySerAspSerTyr TrpArgIleProValAspAspValTyrValGlyGlyThrSerCysAspPheSerAsnLys SerAlaIleIleAspThrGlyThrSerTyrAlaMetLeuProSerSerAspSerLysThr LeuHisSerLeuIleProGlyAlaLysSerSerGlySerTyrHisIleIleProCysAsn ThrThrThrLysLeuGlnValAlaPheSerGlyValAsnTyrThrIleSerProLysAsp TyrValGlyAlaThrSerGlySerGlyCysValSerAsnIleIleSerTyrAspLeuPhe GlyAspAspIleTrpLeuLeuGlyAspThrPheLeuLysAsnValTyrAlaValPheAsp TyrAspGluLeuArgValGlyPheAlaGluArgSerSerAsnThrThrSerAlaSerAsn SerThrSerSerGlyThrSerSerThrSerGlySerThrThrThrGlySerSerThrThr ThrThrSerSerAlaSerSerSerSerSerSerAspAlaGluSerGlySerSerMetThr IleProAlaProGlnTyrPhePheSerAlaLeuAlaIleAlaSerPheMetLeuTrpLeu SEQ ID NO: 13 Organism Glomerella graminicola M1.001 (fungus), 472 aa   1 mggsfrpnya dgpeppfspt kkttlviikt sllipgdgep lkdgalviss kviawvgpqs  61 slpseyadsp hrsytvpylm pglwdchahf ggespnddgg ndpyqvfite hpaasgarlt 121 rgcwlalqrg ytslrdvagl gcevsraied gsivgpnvys sgsglsqlag hgdifslpag 181 dvllnlgvsq itpghfgtha tmivdgvdec rravrlqirr gakcikvmas ggvmsrddnp 241 nyaqfsaael etiveeatrq nrvvaahvhg kagilaaina gvttlehasf adrecidlik 301 ekgivyiatr ivvhlllstg gkglpptvwe kaklvaknhm taykmaiesg vqialgtdtg 361 pgynmatele caveagmsnl eaikaatang plsvggqapk tgqlkvgyea dvigllqnpv 421 edvkvlqkvd nvgwvwkggk lfkgpgvgpw geepgvwedi tgvsrlrctt gy SEQ ID NO: 14 Organism Metarhizium anisopliae ARSEF 23. 485 aa,   1 mtrrviphse saesvddaqh vsgriystgf giavrtgqpq sqdddaetgv kkvfytivmt  61 kllipgdgep ikdaalvvkn kiidwvgrqa dlpneytekp hklhnvpylm pglwdchvhf 121 agsngereae egstglsfla dhpttagarl argcwdaiqr gytsmrdlag fgceiskaie 181 dgviigpniy sagaclsqla ghgdvfalpa gdallnlgla svkagqfgag msclvdgvde 241 crrgvrlqir rgakcikvma sggvlsrddn plyaqfsree ldtivseakr mertvaahvh 301 gkpgilqave agvtsvehvs fadqecidli kdrgtifvgt rtivnlllds kgegmpkkmw 361 ekaklvgths legykkaika gctialgtdt epgfnmaiel qyaveagmss leaikaatan 421 gpltvagqap ltgqlkagye admigvcdnp vedvkvlqkk snigwvwkgg klfkgpgigp 481 wgeel SEQ ID NO: 15 A. oryzae amidohydrolase Mhrmltddrlyrvdadllipgkgdpiphgavvwqcktiryagprsgvptefqgattthvpvvmpgmwdc hihflgataatmnaivdtpqalagarsvpdlhatvmagftsvrevggygcdlakavgegripgpniyss hsaismtaghgdvhgvhrdslldlcahglpltiadgvpecllavrkqlrrgakvikvcasggvvsaidd pqhqefsfeelkaivdeaararrvvaahchgkagimnalragcrtiehgsfldeeavglmkekgailva trsviesglamkdlftpssyqkllavadahrkayqlaisrgvtialgtdqfissdnpmigygrnghevr yavdagltplaaieaatangpltlgyqapqsgqlkegydadiiavrenplenvavlsnsknvthvwrgg mliks SEQ ID NO: 16 Bovine carboxypeptidase A (419 aa). Accession: NP_777175. Sigma product no. C0261.   1 mqgllilsvl lgaalgkedf vghqvlrita adeaevqtvk eledlehlql dfwrgpgqpg  61 spidvrvpfp slqavkvfle ahgiryrimi edvqslldee qeqmfasqsr arstntfnya 121 tyhtldeiyd fmdllvaehp qlvsklqigr syegrpiyvl kfstggsnrp aiwidlgihs 181 rewitqatgv wfakkftedy gqdpsftail dsmdifleiv tnpdgfafth sqnrlwrktr 241 svtssslcvg vdanrnwdag fgkagasssp csetyhgkya nsevevksiv dfvkdhgnfk 301 aflsihsysq lllypygytt qsipdkteln qvaksaveal kslygtsyky gsiittiyqa 361 sggsidwsyn qgikysftfe lrdtgrygfl lpasqiipta getwlgvlti mehtlnnly SEQ ID NO: 17 Carboxypeptidase Y from baker's yeast (Saccharomyces cerevisiae), 532 aa, Accession: EDV11788. Sigma product no. C3888.   1 mkaftsllcg lglsttlaka islqrplgld kdvllqaaek fglnldldhl lkeldsnvld  61 awaqiehlyp nqvmsletst kpkfpeaikt kkdwdfvvkn daienyqlrv nkikdpkilg 121 idpnvtqytg yldvededkh fffwtfesrn dpakdpvilw lnggpgcssl tglffelgps 181 sigpdlkpig npyswnsnat vifldqpvnv gfsysgssgv sntvaagkdv ynflelffdq 241 fpeyvnkgqd fhiagesyag hyipvfasei lshkdrnfnl tsvlignglt dpltqynyye 301 pmacgeggep svlpseecsa medslerclg liescydsqs vwscvpatiy cnnaqlapyq 361 rtgrnvydir kdceggnlcy ptlqdiddyl nqdyvkeavg aevdhyescn fdinrnflfa 421 gdwmkpyhta vtdllnqdlp ilvyagdkdf icnwlgnkaw tdvlpwkyde efasqkvrnw 481 tasitdevag evksykhfty lrvfngghmv pfdvpenals mvnewihggf sl

REFERENCE LIST

-   1 Abrunhosa, L. and Venancio, A. (2007). Isolation and purification     of an enzyme hydrolyzing ochratoxin A from Aspergillus niger.     Biotechnol. Lett. 29: 1909-1914 -   2 Abrunhosa, L., Paterson, R. R. M. and Venancio, A. (2010).     Biodegradation of Ochratoxin A for Food and Feed Decontamination.     Toxins, 2: 1078-1099 -   3 Fraser, J. A., Davis, M. A., and Hynes, M. J. (2001). The     Formamidase Gene of Aspergillus nidulans: Regulation by Nitrogen     Metabolite Repression and Transcriptional Interference by an     Overlapping Upstream Gene. Genetics 157: 19-131 -   4 Jelinek, C. F., Pohland, A. E., and Wood, G. E. (1989). Worldwide     Occurrence of Mycotoxins in Foods and Feeds—An Update. Journal of     the Association of Official Analytical Chemists 72: 223-230 -   5 Li, S., Marquardt, R. R., Frohlich, A. A., Vitti, T. G., and     Crow, G. (1997). Pharmacokinetics of ochratoxin A and its     metabolites in rats. Toxicol. Appl. Pharmacol. 145: 82-90 -   6 Pitout, M. J. (1969). The hydrolysis of ochratoxin A by some     proteolytic enzymes. Biochem. Pharmacol. 18: 485-491 -   7 Stander, M. A., Bornscheuer, U. T., Henke, E., and Steyn, P. S.     (2000). Screening of commercial hydrolases for the degradation of     ochratoxin A. J. Agric. Food Chem. 48: 5736-5739 -   8 Van der Merwe, K. J., Steyn, P. S., and Fourie, L. (1965).     Mycotoxins. II. The constitution of ochratoxins A, B, and C,     metabolites of Aspergillus ochraceus Wilh. J. Chem. Soc. [Perkin 1]     7083-7088 -   9 WHO/FAO (1998). Food irradiation. A Technique for Preserving and     Improving the Safety of Food. World Health Organization in     collaboration with the Food and Agricultural Organization, Geneva -   10 Wang, H (2010). Increased production of aspartic proteases in     filamentous fungal cells. WO 2010014574 (A2) 

The invention claimed is:
 1. An isolated amidase enzyme capable of degrading ochratoxin, wherein the isolated amidase enzyme comprises a polypeptide sequence having a sequence of SEQ ID NO: 3 that has a cleavable tag, or a sequence having at least 70% sequence identity to SEQ ID NO: 3 that has the cleavable tag.
 2. The amidase according to claim 1, wherein the ochratoxin is ochratoxin A.
 3. The amidase according to claim 1, wherein the amidase comprises at least one of the amino acid sequence motifs: 1) x-P-G-x-x-D-x-H-x-H-x-xG; 2) G-x-T; 3) G-x-x-x-G-P; 4) G-H-x-D; 5) D-G-x-x-x-V-x-x-x-x-R-x-x-x-R-x-x-A-x-x-I-K; 6) G-G-V-x-S-x-x-D-x-P; 7) V-x-A-H-x-x-G-x-x-G; 8) H-x-x-x-x-D; or 9) G-V-x-I-x-x-G-T-D;

wherein x is defined as any amino acid.
 4. The amidase according to claim 1, wherein the amidase has a Tim barrel structure including an active site comprising 6 histidine residues, 1 lysine residue and 1 aspartic acid residue, wherein the amino acid residues in the active site correspond to positions H111, H113, H191, K246, H287, H289, H307 and D378 of SEQ ID NO:1 when the tertiary structure of the amidase and SEQ ID NO:1 are compared.
 5. The amidase according to claim 1, wherein the amidase enzyme comprises a polypeptide sequence having at least 75% identity to SEQ ID NO: 3 and which differs from SEQ ID NO: 3 by one or more amino acid additions, deletions or substitutions.
 6. The amidase according to claim 1, wherein the amidase hydrolyzes about at least 100 to about 900 nanomoles ochratoxin A per min per mg protein at pH7.0 and 40° C.
 7. The enzyme according to claim 3, wherein the motifs comprise: 1) x¹-P-G-x²-x³-D-x⁴-H-x⁵-H-x⁶-x⁷-G; 2) G-x⁸-T; 3) G-x⁹-x¹⁰-x¹¹-G-P; 4) G-H-x¹²-D; 5) D-G-x¹³-x¹⁴-x¹⁵-C-x¹⁶-x¹⁷-x¹⁸-x¹⁹-R-x²⁰-x²¹-x²²-R-x²³-x²⁴-A-x²⁵-x²⁶-I-K, 6) G-G-V-x²⁷-S-x²⁸-x²⁹-D-x³⁰-P; 7) V-x³¹-A-H-x³²-x³³-G-x³⁴-x³⁵-G; 8) H-x³⁶-x³⁷-x³⁸-x³⁹-D; or 9) G-V-x⁴⁰-I-x⁴¹-x⁴²-G-T-D;

wherein: X¹=l/m; x²=l/m; x³=w/i; x⁴=c/v/s/a; x⁵=any amino acid; x⁶=f/y/l; x⁷=any amino acid; x⁸=y/f; x⁹=t/a/s/v/r; x¹⁰=i/f/a; x¹¹=any amino acid; x¹²=g/s; x¹³=v/e; x¹⁴=any amino acid; x¹⁵=e/d/g; x¹⁶=any amino acid; x¹⁷=any amino acid; x¹⁸=a/g/t; x¹⁹=v/a; x²⁰=any amino acid; x²¹=q/m/a; x²²=l/i/v; x²³=r/h/c; x²⁴=g/n; x²⁵=k/r/t/e/d; x²⁶=any amino acid; x²⁷=l/m/v/g; x²⁸=any amino acid; x²⁹=any amino acid; x³⁰=any amino acid; x³¹=a/s/h; x³²=c/v/a; x³³=h/q; x³⁴=k/r; x³⁵=any amino acid; x³⁶=g/v/a; x³⁷=s/t/i; x³⁸=y/f/e; x³⁹=l/a/i; x⁴⁰=any amino acid; x⁴¹=a/v; x⁴²=−l/a, wherein x is defined as any amino acid.
 8. The enzyme according to claim 7, wherein the motifs comprise: 1) l/m-P-G-l/m-w-D-c-H-x-H-f-x-G; 2) G-y/f-T; 3) G-t-i-x-G-P; 4) G-H-g-D; 5) D-G-v-x-e-V-x-x-a-v-R-x-q-l-R-r-g-A-k-x-I-K; 6)G-G-V-l/-S-x-x-D-x-P; 7) V-a-A-H-c-h-G-k-x-G; 8) H-g-s-y-l-D; or 9) G-V-x-I-a-l-G-T-D;

wherein x is defined as any amino acid.
 9. A feed or food composition comprising an amidase enzyme capable of degrading ochratoxin, wherein the amidase enzyme comprises a polypeptide sequence having a sequence of SEQ ID NO: 3 or a polypeptide sequence having at least 70% sequence identity to SEQ ID NO: 3, wherein said feed or food composition is selected from the group consisting of: grains, by products from cereals, silage, protein, oils and fats, and minerals and vitamins.
 10. The feed or food composition according to claim 9, wherein the ochratoxin is ochratoxin A.
 11. The feed or food composition according to claim 9, wherein the amidase comprises at least one of the amino acid sequence motifs: 1) x-P-G-x-x-D-x-H-x-H-x-xG; 2) G-x-T; 3) G-x-x-x-G-P; 4) G-H-x-D; 5) D-G-x-x-x-V-x-x-x-x-R-x-x-x-R-x-x-A-x-x-I-K; 6) G-G-V-x-S-x-x-D-x-P; 7) V-x-A-H-x-x-G-x-x-G; 8) H-x-x-x-x-D; or 9) G-V-x-I-x-x-G-T-D;

wherein x is defined as any amino acid.
 12. The feed or food composition according to claim 9, wherein the amidase has a Tim barrel structure including an active site comprising 6 histidine residues, 1 lysine residue and 1 aspartic acid residue, wherein the amino acid residues in the active site correspond to positions H111, H113, H191, K246, H287, H289, H307 and D378 of SEQ ID NO:1 when the tertiary structure of the amidase and SEQ ID NO:1 are compared.
 13. The feed or food composition according to claim 9, wherein the feed or food additive comprises a physiologically acceptable carrier selected from at least one of maltodextrin, limestone (calcium carbonate), cyclodextrin, wheat, wheat bran or a wheat component, rice or rice bran, sucrose, starch, Na₂SO₄, Talc, and PVA and mixtures thereof.
 14. The feed or food additive according to claim 9, wherein the feed or food additive comprises one or more further feed enzyme(s) selected from the group consisting of those involved in protein degradation including carboxypeptidases carboxypeptidase A, carboxypeptidase Y, A. niger aspartic acid proteases of PEPAa, PEPAb, PEPAc and PEPAd, elastase, amino peptidases, pepsin or pepsin-like, trypsin or trypsin-like proteases and bacterial proteases including subtilisin and its variants, and of those involved in starch metabolism, fibre degradation, lipid metabolism, proteins or enzymes involved in glycogen metabolism, amylases, arabinases, arabinofuranosidases, catalases, cellulases, chitinases, chymosin, cutinase, deoxyribonucleases, epimerases, esterases, -galactosidases, glucanases, glucan lysases, endo-glucanases, glucoamylases, glucose oxidases, -glucosidases, including β glucosidase, glucuronidases, hemicellulases, hexose oxidases, hydrolases, invertases, isomerases, lipolytic enzymes, laccases, lyases, mannosidases, oxidases, oxidoreductases, pectate lyases, pectin acetyl esterases, pectin depolymerases, pectin methyl esterases, pectinolytic enzymes, peroxidases, phenoloxidases, phytases, polygalacturonases, proteases, rhamno-galacturonases, ribonucleases, thaumatin, transferases, transport proteins, transglutaminases, xylanases, hexose oxidase (D-hexose: O₂-oxidoreductase, EC 1.1.3.5), or acid phosphatases and combinations thereof.
 15. A feed or food material comprising the feed additive of claim
 9. 16. A feedstuff or foodstuff comprising the food or feed material of claim
 15. 17. The feedstuff according to claim 16, comprising one or more feed materials selected from the group consisting of a) cereals, small grains (wheat, barley, rye, oats and combinations thereof) and/or large grains, maize or sorghum; b) byproducts from cereals, corn gluten meal, Distillers Dried Grain Solubles (DDGS), wheat bran, wheat middlings, wheat shorts, rice bran, rice hulls, oat hulls, palm kernel, and citrus pulp; c) silage; d) protein obtained from soya, sunflower, peanut, lupin, peas, fava beans, cotton, canola, fish meal, dried plasma protein, meat and bone meal, potato protein, whey, copra, sesame; e) oils and fats obtained from vegetable and animal sources; f) minerals and vitamins.
 18. The foodstuff according to claim 16, wherein said foodstuff comprises corn, wheat, barley, sorghum, a cereal product, porridges, noodles, bread or cakes, coffee, cocoa, wine, beer, pulses, spices, dried fruit, grape juice, milk, milk products, cheese, meat or meat products.
 19. The enzyme according to claim 11, wherein the motifs comprise: 1) x¹-P-G-x²-x³-D-x⁴-H-x⁵-H-x⁶-x⁷-G; 2) G-x⁸-T; 3) G-x⁹-x¹⁰-x¹¹-G-P; 4) G-H-x¹²-D; 5) D-G-x¹³-x¹⁴-x¹⁵-C-x¹⁶-x¹⁷-x¹⁸-x¹⁹-R-x²⁰-x²¹-x²²-R-x²³-x²⁴-A-x²⁵-x²⁶-I-K, 6) G-G-V-x²⁷-S-x²⁸-x²⁹-D-x³⁰-P; 7) V-x³¹-A-H-x³²-x³³-G-x³⁴-x³⁵-G; 8) H-x³⁶-x³⁷-x³⁸-x³⁹-D; or 9) G-V-x⁴⁰-I-x⁴¹-x⁴²-G-T-D;

wherein: x¹=1/m; x²=1/m; x³=w/i; x⁴=c/v/s/a; x⁵=any amino acid; x⁶=f/y/l; x⁷=any amino acid; x⁸=y/f; x⁹=t/a/s/v/r; x¹⁰=i/f/a; x¹¹=any amino acid; x¹²=g/s; x¹³=v/e; x¹⁴=any amino acid; x¹⁵=e/d/g; x¹⁶=any amino acid; x¹⁷=any amino acid; x¹⁸=a/g/t; x¹⁹=v/a; x²⁰=any amino acid; x²¹=q/m/a; x²²=l/i/v; x²³=r/h/c; x²⁴=g/n; x²⁵=k/r/t/e/d; x²⁶=any amino acid; x²⁷=l/m/v/g; x²⁸=any amino acid; x²⁹=any amino acid; x³⁰=any amino acid; x³¹=a/s/h; x³²=c/v/a; x³³=h/q; x³⁴=k/r; x³⁵=any amino acid; x³⁶=g/v/a; x³⁷=s/t/i; x³⁸=y/f/e; x³⁹=1/a/i; x⁴⁰=any amino acid; x⁴¹=a/v; x⁴²=−l/a.
 20. A method of making a foodstuff or feedstuff according to claim 16 comprising adding to a food or feed material a food or feed additive that comprises an amidase enzyme capable of degrading ochratoxin.
 21. A method of making a food or feed additive according to claim 16 comprising admixing the amidase with at least one physiologically acceptable carrier.
 22. A method of reducing ochratoxin contamination of a feed or food composition comprising adding to said feed or food composition an ochratoxin degrading amidase enzyme, wherein the ochratoxin degrading amidase enzyme comprises a polypeptide sequence having a sequence of SEQ ID NO: 3 or a polypeptide sequence having at least 70% sequence identity to SEQ ID NO: 3, wherein said feed or food composition is selected from a group consisting of: grains, by products from cereals, silage, protein, oils and fats, and minerals and vitamins.
 23. The method according to claim 22, wherein the amidase comprises at least one of the amino acid sequence motifs: 1) x-P-G-x-x-D-x-H-x-H-x-xG; 2) G-x-T; 3) G-x-x-x-G-P; 4) G-H-x-D; 5) D-G-x-x-x-V-x-x-x-x-R-x-x-x-R-x-x-A-x-x-I-K; 6) G-G-V-x-S-x-x-D-x-P; 7) V-x-A-H-x-x-G-x-x-G; 8) H-x-x-x-x-D; or 9) G-V-x-I-x-x-G-T-D.


24. A recombinant cell or spore encoding an amidase enzyme which degrades ochratoxin, wherein the amidase enzyme comprises a polypeptide sequence having a sequence of SEQ ID NO: 3 that has a cleavable tag, or a sequence having at least 70% sequence identity to SEQ ID NO: 3 that has the cleavable tag.
 25. The cell or spore according to claim 24 wherein the amidase enzyme degrades ochratoxin A.
 26. The cell or spore according to claim 24, wherein the amidase is isolated.
 27. The cell or spore according to claim 24, wherein the amidase comprises at least one of the amino acid sequence motifs: 1) x-P-G-x-x-D-x-H-x-H-x-xG; 2) G-x-T; 3) G-x-x-x-G-P; 4) G-H-x-D; 5) D-G-x-x-x-V-x-x-x-x-R-x-x-x-R-x-x-A-x-x-I-K; 6) G-G-V-x-S-x-x-D-x-P; 7) V-x-A-H-x-x-G-x-x-G; 8) H-x-x-x-x-D; or 9) G-V-x-I-x-x-G-T-D.


28. The cell or spore according to claim 24, wherein the amidase has a Tim barrel structure including an active site comprising 6 histidine residues, 1 lysine residue and 1 aspartic acid residue, wherein the amino acid residues in the active site correspond to positions H111, H113, H191, K246, H287, H289, H307 and D378 of SEQ ID NO:1 when the tertiary structure of the amidase and SEQ ID NO:1 are compared.
 29. The recombinant cell or spore according to claim 24, wherein the amidase is at least 30% pure. 